Julian attended DePauw University in Greencastle, Indiana. The college accepted few African-American students. The segregated nature of the town forced social humiliations. Julian was not allowed to live in the college dormitories and first stayed in an off-campus boarding home, which refused to serve him meals. It took him days before Julian found an establishment where he could eat. He later found work firing the furnace, waiting tables, and doing other odd jobs in a fraternity house; in return, he was allowed to sleep in the attic and eat at the house. Julian graduated from DePauw in 1920 as a Phi Beta Kappa and valedictorian.

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In 1923 he received an Austin Fellowship in Chemistry, which allowed him to attend Harvard University to obtain his M.S. However, worried that Euro-American students would resent being taught by an African-American; Harvard withdrew Julian's teaching assistantship, making it financially impossible for him to complete his Ph.D.

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In 1929, while an instructor at Howard University, Julian received a Rockefeller Foundation fellowship to continue his graduate work at the University of Vienna, where he earned his Ph.D. in 1931.

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After returning from Vienna, Julian taught for one year at Howard University. At Howard, in part due to his position as a department head, Julian became caught up in university politics. At university president Mordecai Wyatt Johnson's request, he goaded white Professor of chemistry, Jacob Shohan (Ph.D. from Harvard, into resigning. In May 1932, Shohan retaliated by releasing to the local African-American newspaper the letters Julian had written to him from Vienna. The letters described "a variety of subjects from wine, pretty Viennese women, music and dances, to chemical experiments and plans for the new chemical building." In the letters, he spoke with familiarity, and with some derision, of specific members of the Howard University faculty, terming one well-known Dean, an "ass".

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Around this same time, Julian also became entangled in an interpersonal conflict with his laboratory assistant, Robert Thompson. Julian had recommended Thompson for dismissal in March 1932. Thompson sued Julian for "alienating the affections of his wife", Anna Roselle Thompson, who Julian would go on to marry in 1935. When Thompson was fired, he too gave the paper intimate and personal letters which Julian had written to him from Vienna. Dr. Julian's letters revealed "how he fooled the [Howard] president into accepting his plans for the chemistry building" and "how he bluffed his good friend into appointing" a professor of Julian's liking. Through the summer of 1932, the Baltimore Afro-American published all of Julian's letters. Eventually, the scandal and accompanying pressure forced Julian to resign.

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At the lowest point in Julian's career, a former mentor threw him a much-needed lifeline. Julian was offered a position to teach at DePauw University in 1932. Julian then helped Josef Pikl, a fellow student at the University of Vienna, to come to the United States to work with him at DePauw. In 1935 Julian and Pikl completed the total synthesis of physostigmine and confirmed the structural formula assigned to it. Robert Robinson of Oxford University in the U.K. had been the first to publish a synthesis of physostigmine, but Julian noticed that the melting point of Robinson's end product was wrong, indicating that he had not created it. When Julian completed his synthesis, the melting point matched the correct one for natural physostigmine from the Calabar bean.

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Julian also extracted stigmasterol which took its name from the West African Calabar Bean that he hoped could serve as raw material for synthesis of human steroidal hormones. In 1934, German chemists had shown that stigmasterol, isolated from soybean oil, could be converted to progesterone by synthetic organic chemistry.

Private sector work: Glidden

In 1936 Julian was denied a professorship at DePauw for racial reasons. DuPont had offered a job to fellow chemist Josef Pikl but declined to hire Julian, despite his superlative qualifications, apologizing that they were "unaware he was a Negro". Meanwhile, Julian had written to the Glidden Company, a supplier of soybean oil products, to request a five-gallon sample of the oil to use as his starting point for the synthesis of human steroidal sex hormones (in part because his wife was suffering from infertility). After receiving the request, Glidden, made a telephone call to Julian, offering him the position of director of research at Glidden's Soya Products Division in Chicago. He was very likely offered the job because he was fluent in German, and Glidden had just purchased a modern solvent extraction plant from Germany for the extraction of vegetable oil from soybeans for paints and other uses.

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Steroids

Julian and his wife, Anna Roselle, had difficulties conceiving.  By 1940, Julian began work on synthesizing human sex hormones progesterone, estrogen, and testosterone from soybean oil. At that time clinicians were discovering many uses for the newly discovered hormones. However, only minute quantities could be extracted from hundreds of pounds of the spinal cords of animals.

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One of Julian's greatest scientific accomplishments resulted from an accident that could have cost him his job. Water leaked into a tank filled with $160,000 worth of pure soybean oil, causing the liquid to spoil and a white sludge to form. Within the sludge, however, lay crystals Julian recognized as stigmasterol, a plant steroid that could be converted into the pregnancy hormone progesterone. Doctors prescribed progesterone to women in an attempt to curb miscarriages, but until Julian's discovery, the drug was simply too costly for many patients to afford. Although he was not the first to convert stigmasterol into progesterone, Julian was the first to produce the hormone affordably and in bulk.

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Julian was able to produce 100 lbs. of mixed soy sterols daily, which had a value of $10,000 ($80,000 today) as sex hormones. The first pound of progesterone he made, valued at $63,500 ($509,000 today),] was shipped to the buyer, Upjohn, in an armored car. Production of other sex hormones soon followed.

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In April, 1949, the Mayo Clinic announced the dramatic effectiveness of cortisone in treating rheumatoid arthritis. The cortisone was produced by Merck at great expense. In September, 1949, Julian announced an improvement in the process of producing cortisone making the process both easier and cheaper.

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In 1953, Julian left Glidden after 18 years, giving up a salary of nearly $50,000 a year (equivalent to $450,000 in 2016), to found his own company, Julian Laboratories, Inc.

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Oak Park and Julian Laboratories

Circa 1950, Julian moved his family to the Chicago suburb of Oak Park, becoming the first African-American family to reside there. Although some residents welcomed them into the community, there was also opposition. Before they even moved in, their home was fire-bombed. Later, after they moved in, the house was attacked with dynamite. The attacks galvanized the community, and a community group was formed to support the Julians. Julian's son later recounted that during these times, he and his father often kept watch over the family's property by sitting in a tree with a shotgun.

Julian won a contract to provide Upjohn with $2 million worth of progesterone (equivalent to $16 million today). To compete against rival firm Syntex, he would have to use the same  as his starting material. Julian used his own money and borrowed from friends to build a processing plant in Mexico, but he could not get a permit from the government to harvest the yams. Abraham Zlotnik, a former University of Vienna classmate whom Julian had helped escape from the holocaust, led a search to find a new source of the yam in Guatemala for the company.

Julian sold his company in 1961 for $2.3 million (equivalent to $18 million in 2016).