Abstract: Objective: To establish a method to determine the concentration of ampicillin and probenecid in human serum by high performance liquid chromatography(HPLC).Methods: (1)Ampicillin was detected by HPLC using norfloxacin as internal standard after sample was sedimented with 0.6% perchloric acid.Higgins^TM C18 column(250 mm×4.6 mm,5 μm) was used,the mobile phase consisted of organic phase(methanol-acetonitrile-isopropanol,100:2:5,v/v) and aqueous phase(0.01 mol/L potassium dihydrogen phosphate,pH 3.5) with a ratio of 23:77,the flow rate was 1.0 ml/min,the detection wavelength was set at 210 nm,and column oven at 30℃.(2)Probenecid was detected by HPLC using valsartan as internal standard after sample was sedimented with acetonitrile.Higgins^TM C18 column(250 mm×4.6 mm,5 μm) was used,the mobile phase consisted of organic phase(methanol-acetonitrile,400:275,v/v) and aqueous phase(acetic acid-water,1:60,v/v) with a ratio of 77:23,the flow rate was 1.0 ml/min,the detection wavelength was set at 280 nm,and column oven at 30℃.Results: Good linearity was obtained within the range of 0.14-11.2 μg/ml for ampicillin(r=0.999 5),and 0.20-16.0 μg/ml for probenecid(r=0.999 1).The mininum detectable concentrations for ampicillin,and probenecid were 0.14 μg/ml and 0.20 μg/ml,respectively.Conclusions: This method is simple,sensitive and specific,which can be applied to study the pharmacokinetics of ampicillin and probenecid.