Abstract: OBJECTIVE To establish a liquid chromatography tandem mass spectrometry(LC-MS/MS) method for the determination of polymyxin B concentration in human plasma, and to use it for clinical drug monitoring of polymyxin B. METHODS The Welch Ultimate LP-C₁₈(100 mm×2.1 mm, 5 μm) column was used to separate the main components of polymyxin B, and an LC-MS/MS with a time program of 4.0 min was established. Acetonitrile was used as precipitator for sample pretreatment of protein precipitation, positive ion scanning and multi-reaction detection mode were used for analyte determination. RESULTS The linear range of total polymyxin B concentration was 100-10 000 ng·mL^-1. The linear coefficients of the main components were both> 0.996. The intra-assay and inter-assay precisions were both ≤ 6%, and the accuracy was 91.88%-114.02%. The in vivo exposure of three patients using polymyxin B, AUC_{0-24 h} was 39.73, 64.15 and 107.75 mg·L^-1·h^-1, respectively. CONCLUSION This study has established a robust LC-MS/MS method for the determination of the total concentration of polymyxin B in human plasma, with good sensitivity and specificity, and has been successfully used for clinical monitoring the concentration of polymyxin B.