Abstract: OBJECTIVE To establish a RP-HPLC method for simultaneous determination of zinc gluconate and lysine hydrochloride in lysine hydrochloride and zinc gluconate tablet. METHODS Reverse phase ODS column was used as the stationary phase and 0.05 mol·L-1 KH2PO4 (pH=3.00) was used as mobile phase with 210 nm for detection wavelength. RESULTS The calibration curve was linear over the range of 0.0453-5.80 mmol·L-1 for zinc gluconate and 0.133-17.0 mmol·L-1 for lysine hydrochloride.The recovery was 98.8%～101.9%. The coefficients of variation for within day and between days were all less than 2.0%(n=5). CONCLUSION The method could be easily employed with high precision and accuracy for simultaneous determination of zinc gluconate and lysine hydrochloride in lysine hydrochloride and zinc gluconate tablet without prior separation, and could be used for the quality control in production process of the tablet.