OBJECTIVE: To establish a rapid, simple and sensitive HPLC method for determination of fentanyl (Fen) plasma concentration and provide scientific analysis for the studies of the pharmacokinetics of fentanyl in clinical anesthesia. METHOD: Fen was separated on μ-BondapakC₁₈ column with a mobile phase of water (containing 0.005 mol·L^-1 C₈H₁₇O₃Sna and 0.01 mol·L^-1H₃PO₄)acetonitrile (69∶31) and detected at 220 nm. Internal standard was alfentanil. RESULTS: The separation of Fen and alfentanil was proved to be highly satisfactory. The mean recovery of Fen was 99.97%. The intraday and interday RSD were all less than 8%. The assay linearity was determined over the range of 10 ng·ml^-1～200 ng·ml^-1in plasma (r=0.999 6). The minimum detection of Fen was 5 ng·ml^-1. CONCLUSION: This method possesses high accuracy and precision, and is sensitive and specific. The method could be used in the determination of plasma concentration in clinics.