This study explored the short‐term effects of vitamin K2 (VK2) supplementation on biochemical parameters (vitamin D, vitamin E, vitamin A, alkaline phosphatase, calcium, phosphorus (P), magnesium, metallothionein, triglycerides, cholesterol, high‐density lipoprotein (HDL), low‐density lipoprotein (LDL), and lipoprotein fractions (albumin, HDL, very low‐density lipoprotein (VLDL), LDL, and chylomicrons). A short‐term experiment (24 h, six probands) was performed to track changes in VK2 levels after a single‐dose intake (360 µg/day). Liquid chromatography–tandem mass spectrometry was used to monitor vitamin K levels (menaquinone‐4 (MK‐4), menaquinone‐7 (MK‐7), and vitamin K1 [VK1]) with a limit of detection of 1.9 pg/mL for VK1 and 3.8 pg/mL for the two forms of VK2. Results showed that MK‐7 levels significantly increased within 2–6 h post‐administration and then gradually declined. MK‐4 levels were initially low, showing a slight increase, whereas VK1 levels rose initially and then decreased. Biochemical analyses indicated no significant changes in sodium, chloride, potassium, calcium, magnesium, albumin, or total protein levels. A transient increase in P was observed, peaking at 12 h before returning to baseline. Agarose gel electrophoresis of lipoprotein fractions revealed distinct chylomicron bands and variations in VLDL and HDL mobility, influenced by dietary lipids and VK2 supplementation. These findings suggest effective absorption and metabolism of MK‐7 with potential implications for bone metabolism and cardiovascular health.