Technical Abstract: There is increased interest in accurately assessing the total dietary intake of vitamins from all sources, including foods and dietary supplements. Isotope dilution mass spectrometry (IDMS) can be a definitive analytical method for very accurate concentration determinations. A liquid chromatographic (LC) IDMS method for determination of multiple water soluble vitamins in dietary supplement tablets has been developed. As part of a collaborative characterization of the NIST Standard Reference Material 3280: Multivitamin/Multimineral Tablets, isotope-labeled (13C and/or 2H) B-vitamins (B1-thiamine, B6-pyridoxine, nicotinamide, and pantothenic acid) were obtained from commercial sources, with support of the Office of Dietary Supplements (NIH). Our LC/IDMS method (developed in part to help assign values to SRM 3280) uses a C18 reverse phase column, an Agilent 1100 HPLC system, and a Quattro Micro triple-quad mass spectrometer (MS). B-vitamin determination was achieved using a gradient LC profile combined with MS/MS detection in Multiple Reaction Monitoring mode. Stock solutions of the isotope-labeled vitamins were calibrated against USP standard solutions. The SRM tablets, with added amounts of the four isotope-labeled B-vitamins, were extracted and analyzed in a single LC run. Unknown vitamin concentrations were calculated by comparing the ratio of the integrated LC peak at the different masses of the unlabeled and labeled vitamins.