BACKGROUND Given the growing interest in the health benefits of vitamin K, there is great need for development of new high-throughput methods for quantitative determination of vitamin K in plasma. We describe a simple and rapid method for measurement of plasma vitamin K1 (phylloquinone [PK]) and K2 (menaquinones [MK]-4 and -7). Furthermore, we investigated the association of fasting plasma vitamin K with functional vitamin K insufficiency in renal transplant recipients (RTR). METHODS We used HPLC-tandem mass spectrometry with atmospheric pressure chemical ionization for measurement of plasma PK, MK-4, and MK-7. Solid-phase extraction was used for sample clean-up. Mass spectrometric detection was performed in multiple reaction monitoring mode. Functional vitamin K insufficiency was defined as plasma desphospho-uncarboxylated matrix Gla protein (dp-ucMGP) >500 pmol/L. RESULTS Lower limits of quantitation were 0.14 nmol/L for PK and MK-4 and 4.40 nmol/L for MK-7. Linearity up to 15 nmol/L was excellent. Mean recoveries were >92%. Fasting plasma PK concentration was associated with recent PK intake (ρ=0.41, p=0.002) and with plasma MK-4 (ρ=0.49, p<0.001). Plasma PK (ρ=0.38, p=0.003) and MK-4 (ρ=0.46, p<0.001) were strongly correlated with plasma triglyceride concentrations. Furthermore, we found that MK-4-triglyceride ratio, but not PK-triglyceride ratio, was significantly associated with functional vitamin K insufficiency (OR 0.22 [0.07-0.70], p=0.01) in RTR. CONCLUSIONS The developed rapid and easy-to-use LC-MS/MS method for quantitative determination of PK, MK-4, and MK-7 in human plasma may be a good alternative for the labor-intensive and time-consuming LC-MS/MS methods and enables a higher sample throughput.