A reversed-phase, isocratic high performance liquid chromatographic method with acid mobile phase can separate sulbactam and cafoperazone within 12 minutes. Column packed with Hypersil ODS2(250 mm x 4.6 mm i.d., 5 microns) was manufactured by Dalian Elite Company. Mobile phase is composed of water (adjusted to pH 4.0 with 1% phosphoric acid) and acetonitrile (80:20, V/V). The detection was performed at 210 nm and the injection volume was 2 microL. Cefoperazone and sulbactam have good linearity in the ranges of 100 mg/L to 800 mg/L and 100 mg/L to 1,000 mg/L with the correlation coefficients of 0.9991 and 0.9997 respectively. This method is easily to be operated and can be applied for manufacturing and medicinal study.