Objective: Hyperandrogenism is one of the diagnostic criteria for the polycystic ovary syndrome (PCOS) despite no agreed definition of hyperandrogenism. In part, this is due to the quality of testosterone immunoassays. We have developed liquid chromatography-tandem mass spectrometry methods for analysing testosterone and androstenedione (Ad) to study their reference ranges and diagnostic utility in PCOS. Design, setting and subjects: A consecutive series of 122 women attending a reproductive medicine clinic. Methods: Blood samples were taken during the early follicular phase for measurement of LH, FSH, oestradiol, Ad, testosterone and sex hormone-binding globulin (SHBG). Retrospective case note analysis was used to determine the clinical features and ultrasound findings. Results: The incidence of PCOS was 13.9%. The reference interval for testosterone was !1.8 nmol/l and for Ad was 1.4-7.4 nmol/l. There were significant differences in total testosterone (PZ0.001), Ad (P!0.05) and free androgen index (FAI; P!0.0001) between the women with and without PCOS. Diagnostic performance using receiver operator characteristic plots showed area under the curve (AUC) for FAI 0.81, testosterone 0.75 and Ad 0.66. The AUC for the LH:FSH ratio was 0.72. Conclusions: Our analysis of a consecutive series of women attending a reproductive clinic has provided an appropriate series on which to construct reference ranges for key androgens in women. Secondly, it has allowed us to conclude that early follicular serum testosterone measured using tandem mass spectrometry, FAI and the LH:FSH ratio are valuable laboratory tests in the diagnosis of PCOS.