OBJECTIVE: To compare the calculated and the rapid equilibrium dialyzed (ED) human serum free testosterone (FT) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to explore their performances in polycystic ovary syndrome (PCOS) diagnosis. METHODS: A rapid ED LC-MS/MS method for serum FT was established and validated for linearity, lower limit of the measuring interval (LLMI), imprecision, trueness, stability, dilution, matrix specificity and carryover. The validated ED LC-MS/MS (ED-FT) was compared with calculated LC-MS/MS method from Vermeulen's formula (cFT) for FT measurement in 139 PCOS patients and 100 healthy controls. The performances of total testosterone (TT), ED-FT and cFT by LC-MS/MS in PCOS diagnosis were investigated with the same cohorts. RESULTS: The linearity range of ED-FT was 1.74---890 pmol/L, with a LLMI at 1.74 pmol/L. The intra-assay and inter-assay imprecision were <
3.8 % and <
5.9 %. The trueness was acceptable with recoveries of 92.9 % - 108.2 %. The equilibrium dialysis time was 4 h. The two FT methods displayed systematic and proportional differences and cFT showed significant positive deviations compared to ED-FT. Receiver operating characteristic curve analysis proved that ED-FT outperformanced in PCOS diagnosis with the an area under the curve at 0.973, sensitivity of 89.93 % and specificity of 96.00 %. CONCLUSIONS: This study established a rapid, accurate and sensitive ED LC-MS/MS method for serum FT. ED-FT is optimal compared to TT and cFT by LC-MS/MS in PCOS diagnosis.