A new method for the liquid chromatographic-fluorescence determination of serum vitamin K1 is described using reduction of the K-quinone to the fluorescent K-hydroquinone. The reduction reaction occurs "on-line" in the LC system using a catalytic reducer column and an alcohol mobile phase as reductant. A procedure for serum determination utilizes a liquid-liquid serum lipid extraction followed by normal-phase fractionation on a solid-phase extraction cartridge. The final measurement uses a reversed-phase (C18) separation with a ethanol-methanol mobile phase and provides a detection limit of approximately 20 pg/ml.