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Objective: To develope an HPLC-MS/MS method for the determination of four water-soluble vitamins (B1,B2,B6 and B9) in human serum and compare with Electrochemical method(EC) clinically used for routine vitamin determination in patients.Methods: Separation was achieved at 30℃ on a Venusil XBP-C18 column (150×4.6 mm,3 μm,100A),and eluted with the mobile phase of 5 mM ammonium acetate :methanol (40∶60,v/v) at a flow rate of 0.5 mL/min,and with a run time of 6 minutes.