How well does multi-analyte steroid mass spectrometry (MS) profiling classify women with and without polycystic ovary syndrome (PCOS)? Our liquid chromatography MS (LC-MS) steroid profiling only minimally improves discrimination of women with and without PCOS compared with a direct testosterone immunoassay (T_IA) and the free androgen index (FAI). Blood testosterone measured by direct (non-extraction) immunoassay overlaps between women with and without PCOS. Multi-analyte MS provides greater specificity and accuracy for steroid measurement so might improve the classification. An observational, cross-sectional study of women with PCOS (n = 152) defined by Rotterdam criteria and matched non-PCOS (n = 45) control women was conducted. Serum steroid profiles of testosterone (T), dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), androstenedione (A Serum testosterone by immunoassay demonstrated levels more than 100% higher than that measured by LC-MS. Compared with the controls, women with PCOS had higher serum T, DHEA, A The study cohort is limited in size and only unconjugated steroids were measured. Multi-analyte steroid profiling of unconjugated circulating steroids provides only limited improvement on direct T_IA in classifying women with and without PCOS. None. N/A.