Abstract A gradient high-performance liquid chromatographic (HPLC) procedure has been developed for the determination of microgram amounts of cefoperazone in human serum and urine. The method employes a μ Bondapak C 18 column and gradient elution with two mobile phases. Excellent separation of the drug from potential degradation products as well as from representative penicillins (sodium ampicillin, sodium methicillin, potassium penicillin G) and aminoglycosides (tobramycin, gentamicin, kanamycin) has been demostrated. Coefficients of variation of 7.3% or less were obtained for 25–100 μg/ml cefoperazone in both serum and urine. Average recoveries of the drug from spiked serum and urine samples corresponded to 97.6% and 98.6%, respectively. Amounts as low as 1 μg cefoperazone per ml of sample can be estimated using sample volumes corresponding to 0.1 ml serum or 1 ml urine. Good correlation between the HPLC assay and microbiological cylinder—plate assay employing Micrococcus luteus ATCC 9341 has been demostrated for human serum and urine of patients treated with cefoperazone. While the microbiological method is less time-consuming, it lacks specificity in the presence of other antibiotics. The HPLC method can be used to analyze cefoperazone in the presence of penicillins and aminoglycosides which can potentially be co-administered with cefoperazone.