Sex hormones are steroid compounds essential for puberty and pregnancy, yet their structures were unknown in the 1930s. By purifying tens of thousands of liters of human urine and animal ovaries, Butenandt isolated estrone, androsterone and progesterone, then established their molecular formulas and stereochemistry. His work enabled therapy for hormone deficiencies, the development of oral contraceptives, and new hormone-based cancer treatments.

Using chromatography and repeated recrystallization, Butenandt crystallized microgram quantities of steroids. By combining UV spectroscopy, elemental analysis and partial synthesis, he elucidated the skeletons and functional group patterns of estrone (C18 aromatic steroid), androsterone (C19 androgen) and progesterone (C21 luteal hormone). His achievements provided starting materials for total steroid synthesis (e.g., Tihanyi, Ruzicka/La Roche) and gave endocrinology a firm chemical basis. They also triggered advances in quantitative hormone assays, receptor research and modern molecular endocrinology and medicinal chemistry.

Butenandt employed large-scale extraction (≈50 000 L urine) followed by counter-current distribution and fractional crystallization to obtain milligram purities of estrone, androsterone and progesterone. Structural assignment relied on oxidative degradation (e.g., estrone → oestratriene carboxylic acids), dehydrogenation with SeO2, and correlation with cholesterol. His stereochemical proofs pre-dated X-ray crystallography and used optical rotatory dispersion and targeted hydrogenation. The work established the 1,3,5-(10)-estratriene core and clarified C-17 oxidation states, thus laying groundwork for Wieland–Miescher ketone-based syntheses and contraceptive steroid industry. It also spurred receptor isolation and structure–activity relationship studies that underpin present-day endocrine oncology.