OBJECTIVE: To develop a new HPLC method for the isolation and determination of ampicillin and its dergadation products. METHODS: Chromatographed conditions included column:NovaPak C₁₈;mobile phase:0.02 mol·L^-1monobasic sodium phosphate (NaH₂PO₄) solution (pH4.5)acetonitrile (95∶5);detection wavelengths at 230 nm. RESULTS: There was a good linear relationship over the range of 0.24～1.20 mg·ml^-1,r=0.9991.The average recovery was 100.6% with RSD0.47%(n=5).The RSD for repetition was 0.06%(n=5)，ampicillin and its degradation products were isolated on base line. CONCLUSION: The method was simple,rapid,accurate and can be used to assay ampicillin and its degradation prouducts.