A highly reproducible ion-pair reverse-phase high performance liquid chromatographic assay for cefmenoxime in human serum has been developed. A simple sample cleanup procedure is employed. Cefoxitin is the internal standard and separation is achieved using a C-18 μ-Bondapak column. The mobile phase consists of 20% acetonitrile and 80% 0.05 M ammonium acetate buffer containing 0.005 M tetrabutylammonium hydrogen sulphate as the ion-pair agent. Samples are quantitated by UV detector at 254 nm and 0.02 aufs with an assay sensitivity of 0.625 μg/ml. The method has been successfully applied in a clinical setting.