A stability-indicating high pressure liquid chromatographic method was developed to determine the stability of water-soluble vitamins in total parenteral nutrition mixtures. Folic acid and thiamine were determined by direct injection and precolumn enrichment, followed by UV detection. Nicotinamide and pyridoxine were determined simultaneously without sample pretreatment by UV detection of nicotinamide and fluorescence detection of pyridoxine. Riboflavin 5'-phosphate was also determined without elaborate sample handling and by fluorescence detection. Ascorbic acid was determined as the sum of ascorbic acid and dehydroascorbic acid. After derivatization to a quinoxaline the latter substance was determined by direct injection and fluorescence detection.