Abstract: OBJECTIVE To establish an HPLC method for the determination of cefoselis in human plasma. METHODS With metronidazole as the internal standard, the separation of sample was carried out on a ZORBAX Extend C₁₈ column (4.6 mm×250 mm, 5 μm) with the mobile phase consisted of phosphate buffer (pH 6.5)-acetonitrile (94.5∶5.5) at a flow rate of 1.0 mL·min^-1. The detection wavelength was 254 nm, injected sample volume was 20 μL, and the column temperature was set at 25 ℃. RESULTS The linear range of cefoselis was 1-200 mg·L^-1 (r=0.999 9) with the lower limit of quantization of 1 mg·L^-1. The average methodological recovery was 99.8%(RSD=1.53%). The inter-day RSD and intra-day RSD were all less than 3%. CONCLUSION The method is simple, sensitive, rapid, and accurate, and can be used for clinical pharmacokinetic and bioavailability study of cefoselis.