Technical Abstract: The purpose of this study was to develop a Single-Lab Validated Method using high-performance liquid chromatography (HPLC) with different detectors (diode array detector - DAD, fluorescence detector - FLD, and mass spectrometer - MS) for determination of seven B-complex vitamins (B1 - thiamin, B2 – riboflavin, B3 – nicotinamide, B6 - pyridoxine, B9 - folic acid, pantothenic acid, and biotin) and vitamin C in multi-vitamin/multi-mineral dietary supplements. The method involves the use of a reversed phase C18 column (4 µm, 250 mm × 2.0 mm id) and a gradient mobile phase profile. Gradient elution was performed at a flow rate of 0.25 ml/min. After a 5-min isocratic elution at 100% A (0.1% formic acid in water), a linear gradient to 50% A and 50% B (0.1% formic acid in acetonitrile) at 15 min and then to 5% A and 95% B at 17 min was employed. Detection was performed with a DAD as well as either a FLD or a triple-quad MS in the multiple reaction monitoring (MRM) mode. Single Lab Validation was carried out using a standard reference material (SRM) 3280 – Multi-vitamin/Multi-mineral Tablets, being developed with the National Institute of Standards and Technology (NIST), with support by the Office of Dietary Supplements of the National Institutes of Health (NIH). Phosphate buffer (10 mM, pH 2.0) extracts of the NIST SRM 3280 were analyzed by the LC/DAD/FLD/MS method. Following extraction the method does not require any sample clean-up/pre-concentration steps except centrifugation and filtration.