Abstract: A method based on in situ formed ionic liquids microextraction-ultra performance liquid chromatography(ISFILM-UPLC)was established for the determination of vancomycin in human serum. The samples were pretreated by vortex and centrifugation. ［C₆MIM］［Br］ and NaPF₆ were applied as the extracting agent, and Metronidazole as the internal standard. Methanol was applied to redissolve the precipitate. The separation was carried out on a BEH C₁₈ column, and the mobile phase consisted of methanol and 0. 05 mol/L monopotassium phosphate solution. The flow rate was 0. 2 mL/min, the injection volume was 5 μL, and the column temperature was 18 °C. The key parameters of this method such as the specificity, accuracy and precision were validated. This method showed good correlation with the immunoassay. It shows great potential for the application in clinical practice.