Abstract: Objective To develop a new high-performance liquid chromatographic (HLPC) method for determination of propofol in human serum. Methods Human serum samples were precipitated with 20% perchloric acid and centrifuged to obtain 50 μl of the supernatant for analysis by HPLC coupled with fluorescence detection. The analysis was performed with a C18 reversed-phase column using a acetonitrile-water (90∶10) phase delivered at 1.0 ml/min, with the excitation wavelength of 276 nm and emission wavelength of 310 nm. Results The calibration curves were linear (r=0.997 5) within the concentration range of 0.05-10 μg/ml, the limit of propofol quantification was 50 ng/ml and the intra- and inter-day precisions were between ±15%. Conclusion The method is accurate, sensitive and simple for propofol determination in clinical anesthesia.