Objective:To improve the determination method for penicillin V potassium tablets. Methods:An Inertis ODS-SP C18 column (150 mm × 4. 6 mm, 5 μm) was used. The mobile phase consisted of A and B(60∶40) [ A: pH 3. 5 phosphate buffer(0. 5 mol·L-1 potassium dihydrogen phosphate solution, adjusting pH to 3. 5 with phosphoric acid)-acetonitrile-water (10∶30∶60);B:pH 3. 5 phosphate buffer-acetonitrile-water (10∶55∶35)]. The flow rate was 1. 0 ml·min-1, the detection wavelength was 268nm, the column temperature was room temperature and the injection volume was 10 μl. Results:The linear range of penicillin V potassium was 0. 032 9-0. 821 6 mg·ml-1(r=0. 999 9, and the average recovery was 99. 9%( RSD=0. 6%, n=9). Conclusion:The method is reproducible and accurate, and can be used in the content determination of penicillin V potassium tablets and capsules.