The present study describes HPLC/UV method with varying conditions (mobile phases content, flow rates, solvents) for quantitation of water-soluble vitamins (WSV) in supplement mixtures. The method was optimized in respects of analytical and chromatographic parameters such as retention time, symmetry factors, column efficiency as number of theoretical plates, capacity factors, specificity, repeatability, LoD, LoQ and linearity. The performance of these method distinguished with excellent application in the assay tests of WSVs in supplement mixtures containing Thiamine, Riboflavine, Pyridoxine, Niacinamide and Folic acid.