Abstract: Objective To establish a high performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) method for determination of warfarin enantiomers in human plasma. Methods Warfarin enantiomers were extracted with ethyl acetate. The HPLC-MS/MS method used naproxen as internal standard, with methanol∶water∶formic acid=85∶15∶0.05 as mobile phase, at a flow rate of 0.18 mL/min. R-warfain, S-warfarin and internal standard (IS) were separated on column MS Chiral?MS-OD (50×2.1 mm, 3 μm). Warfarin enantiomers were protonated with electroapry ionization (ESI) in negative electron ionization mode. The ion pairs being detected were (m/z) 307.2→160.9 (R-warfain and S-warafrin) and (m/z) 228.9→185.1 (IS). Results The within-run precision relative standard deviations (RSD) and between-run precision RSDof R-warfarin were 3.2%-5.8% and 2.5%-5.1%, respectively. The method recoveries and extraction recoveries of R-warfarin were (96.1±5.6)%-(105.4±4.7)% and 80.7%-84.4%, respectively. The matrix effect RSD was less than 10%. The within-run precision RSD and between-run precision RSDof S-warfarin were 3.7%-5.2% and 3.2%-4.8%, respectively. The method recoveries and extraction recoveries of S-warfarin were (98.3±5.1)%-(103.7±3.8)% and 81.3%-84.6%, respectively. The limit of quantification was 0.1 μg/mL for both analytes. Conclusion This new method is fully validated with satisfactory accuracy and adequate reproducibility. Therefore, it can be applied for separating and detecting plasma warfarin enantiomers.