Objective To establish a convenient, reliable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of warfarin in plasma of patients undergoing cardiac valve replacement surgery. Methods An Agilent G6410A triple quadrupole mass spectrometer with positive ion mode was applied to carry out all experiments. Nitrendipine was used as internal standard. The chromatographic separation was achieved on an Agilent Poroshell 120 Phenyl Hexyl column (2.1 mm×50 mm, 2.7 μm) with isocratic elution. The mobile phase contains 0.2% formic acid and 1% acetonitrile in water (A) and 0.2% formic acid in acetonitrile (B), with volume ratio being 60:40; flow rate was 0.3 mL/min; injection volume was 10 μL; column temperature was 25℃; and analytical time for each sample was 4 min. The precursor ion and the product ion used for quantitative analysis were:warfarin mass-to-charge ratio (m/z) 309→163, nitrendipine m/z 361.2→315.1. Results The linear concentration ranges of free warfarin and total warfarin were 5-500 ng/mL and 50-5 000 ng/mL, respectively, with the correlation coefficient (r) both over 0.990. The relative standard deviation (RSD) values of the lowest limit of quantification were less than 15%. The RSD values of the inter- and intra-day precision were less than 15%. The recoveries of free warfarin and total warfarin by protein precipitation were 78.34%-91.59% and 87.33%-89.99%, respectively, and the matrix effect was 87.50%-104.87%. All the items required in pharmacopeia for methodology validation were acceptable. Conclusion This established LC-MS/MS method can be used to determine the concentrations of free and total warfarin, providing a reference for individualized clinical treatment.