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Polymyxin B (PB) is an antibiotic consisting of a cyclic heptapeptide and a tripeptide side chain used in treatment of infections caused by Gram-negative bacteria. Commercial formulations of PB contain multiple structurally related components with major constituents of PB1, PB2, PB3 and ile-PB1. To understand the pharmacokinetics of these major components, we have developed and validated a LC–MS/MS method to quantify PB1, PB2, PB3 and ile-PB1 in human plasma. PB was extracted from plasma by protein precipitation using trichloroacetic acid followed by chromatographic separation on Zorbax Bonus-RP column (100 mm × 2.1 mm, 1.8 μm) using stepwise gradient elution of water containing 0.1% of formic acid and 0.1% of trichloroacetic acid (mobile phase A) and 90% acetonitrile with 0.1% formic acid (mobile phase B). Despite of structural similarities, these PBs were completely resolved in the analytical run time of 6.5 min. Detection and quantification of PBs were performed by selected reaction monitoring (SRM) under positive ionization mode in the mass spectrometer. Separation of PB1 and ile-PB1, as well as PB2 and PB3, before quantification is crucial because they are structural isomers detected based the same SRM. Excellent linearity was achieved (r2 > 0.99) in the calibration curves of PB. The developed method was accurate (95.3–111.7%) and precise (CV < 5.1%). Recovery of PB from the plasma extraction was between 53 and 76% and reproducible (CV < 4.5%). Matrix effect was not observed by post-column infusion of PB in the mass spectrometer. This methodology has been successfully applied to clinical study of patients dosed with intravenous infusions of PB.We developed and validated a LC–MS/MS method to quantify four major polymyxin B.The method passed FDA guidance on bioanalytical method development.We applied the method in a pharmacokinetic study of patients with infections.