Pyridoxine (vitamin B6) is an essential vitamin which plays a crucial role in the metabolism of amino acids and lipids, but also involved in immune responses and alleviating oxidative stress. There are two other principle forms of vitamin B6, namely, pyridoxal and pyridoxamine. Intracellularly vitamin B6 is converted to its bioactive 5′-phosphate form, pyridoxal 5′-phosphate (PLP), which is essential for many organisms and serves as versatile coenzymes in more than 100 enzymatic reactions. Deficiencies in this vitamin generally lead to the development of inflammatory reaction and major chronic diseases. The following chapter describes a simple and reproducible method for the analysis of pyridoxine via high-performance liquid chromatography utilizing fluorescence detection.