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Human milk is the only source of vitamin K for exclusively breastfed neonates. This vitamin is crucial both for blood coagulation (vitamin K 1 ) and for the normal neurological and skeletal development of the foetus and newborn (vitamin K 2 ). Since vitamin K is ubiquitous in foods, deficiency is not common in adults, but plasma levels and hepatic storage are very low at birth. In light of the importance of this valuable micronutrient, a non-invasive method for verifying that exclusively breastfed infants are receiving an adequate supply of the vitamin is clearly a topic of great significance. In spite of this, the determination of the several vitamin K homologues in human milk has still not been completely elucidated. This paper presents an HPLC–MS/MS method for the simultaneous determination of phylloquinone, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) in human milk after a simple and effective isolation procedure. Overnight cold saponification and extraction of the analytes with hexane provided yields above 75%. This procedure, combined with high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), made it possible to achieve limits of detection (LODs) below 0.8 ng/mL. After a complete validation study, the method was applied to measure vitamin K congeners in several human milk samples; results showed vitamin K 1 concentrations comparable with those reported in the literature. In addition, this is the first study performed for the determination of MK-4 and MK-7 in the maternal milk of Italian women.