Warfarin exerts its anticoagulation activity by blocking the vitamin K-epoxide cycle. A quantitative understanding of how warfarin and related genes interact with the vitamin K-epoxide cycle and the associated change of coagulation activity in the human body may help study the pharmacodynamics of warfarin. The plasma concentration of vitamin K1 (VK1) and vitamin K1 2,3-epoxide (VK1O) could reflect the status of vitamin K-epoxide cycle. However, their determination is a challenging task due to their extremely low concentrations in human plasma and the severe interferences caused by co-extracted lipids. In this study, we developed an LC-APCI-MS/MS method for the simultaneous determination of VK1 and VK1O in human plasma using stable deuterium-labeled vitamin K1 (vitamin K1-d7) as the internal standard (IS). Plasma samples were prepared through protein denaturation followed by one-step liquid extraction with cyclohexane. Chromatographic separation of analytes from isobaric interferences and endogenous ion suppressor was performed on a Synergi Hydro-RP column (150 mm × 4.6 mm, 4 μm) under the reversed-phase condition with isocratic elution. The selective reaction monitoring (SRM) transitions were chosen as m/z = 451.5→187.3 for VK1, m/z = 467.5→161.2 for VK1O, and m/z = 458.6→194.3 for IS in APCI positive mode. The assay was linear in the range of 100–10,000 pg/mL for the two analytes and achieved considerable extraction recoveries (87.8–93.3%, 91.0–96.9%, and 92.0% for VK1, VK1O, and IS, respectively), negligible matrix effects (93.6–96.0%, 96.3–100.1%, and 95.5%), and high selectivity with a small sample volume requirement (0.2 mL) and short run time (15 min). The validated method was successfully applied in a clinical pharmacodynamic study of warfarin, and the clotting activity was found to be negatively correlated with the plasma concentration ratio of VK1O to VK1.