Polymyxin B (PB) is a dose-dependent drug used to treat multidrug-resistantgram-negative bacteria, for which a suitable method is needed to determine clinical samples. A simple, economical, and efficient high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method was developed and validated for polymyxin B1 (PB1), polymyxin B1-Ile (PB1-I), polymyxin B2 (PB2), and polymyxin B3 (PB3) in human plasma. Chromatographic column was Waters BEH C18 column (2.1 × 50 mm, 1.7 μm). Phase A was water with 0.2% formic acid (FA), and phase B was acetonitrile containing 0.2% FA. The elution method is gradient elutio. The total analysis time was 5 min. The pretreatment method involved protein precipitation using acetonitrile containing 0.2% trifluoroacetic acid and 0.1% FA as the precipitant. The recovery rate was 92–99%. The total quantity of PB1 and PB1-I was measured in the linear range of 100–8000 ng/mL. Simultaneously, the total amounts of PB2 and PB3 were measured in the linear range of 11.9–948.5 ng/mL. This validated method was successfully applied to the pharmacokinetics of PB in critically ill patients.