To measure the enzymatic activity of pyridoxine kinase (EC 2.7.1.35) when pyridoxine or pyridoxamine are the substrates an additional enzymatic step is usually required. The products of the kinase activity, pyridoxine 5'-phosphate or pyridoxamine 5'-phosphate, are oxidized, enzymatically, to pyridoxal 5'-phosphate (co-enzyme) which is then measured either spectrophotometrically or using apo-enzymes. In this report the enzymatic activity of pyridoxine kinase, in crude biological extracts, is assayed by a simple high-performance liquid chromatographic method which determines the amount of pyridoxine 5'-phosphate formed when pyridoxine is the substrate. The same method could be used when pyridoxamine is the substrate.