Growing evidence of vitamin K's importance in human health beyond blood coagulation and bone health necessitates its further research. A method involving extraction, lipase treatment, clean-up, and detection and quantification by LC-ESI-MS/MS of phylloquinone (PK), menaquinone-4 (MK-4), menaquinone-7 (MK-7) and menaquinone-9 (MK-9) was developed, and single-laboratory validated. The matrices included in the validation were hazelnut, cheese, broccoli, and pork. The LC-method runtime was 9 min. The LOQ for PK, MK-4 and MK-7 was 0.5 µg/100 g food, while for MK-9 it was 2.5 µg/100 g food. The intra- and inter-day precision was <15% for endogenous and spiked levels, except for low content at 4 times the LOQ. Trueness was assessed to be in the range 94-125% for spiking at levels approximately 4 and 10 times LOQ. It is further shown that deuterium labelled MK-7 can be used as an internal standard for MK-9.