AbstractTo facilitate clinical therapeutic drug monitoring (TDM) of polymyxin B (PB) and polymyxin E (PE), we developed and validated a simple LC–MS/MS method for simultaneous determination of PB (including polymyxin B1 (PB1), polymyxin B2 (PB2), polymyxin B3 (PB3) and isoleucine‐polymyxin B1 (ile‐PB1)) and PE (including polymyxin E1 (PE1) and polymyxin E2 (PE2)) in human plasma. PB or PE was extracted from 20.0 μL plasma using a 5% (v/v) formic acid acetonitrile solution and separated on a BEH‐C18 column (2.1 × 100 mm, 1.7 μm) with a mobile phase consisting of 0.8% formic acid aqueous solution and 0.2% formic acid acetonitrile solution. Gradient elution was performed over 5.5 min at a flow rate of 0.250 mL/min. Quantitative analysis was conducted in positive ion scanning mode by electrospray ionization and multiple reaction monitoring. The method validation was conducted based on bioanalytical method validation guidance, including specificity, calibration curve, precision, accuracy, recovery, matrix effect, stability and dilution integrity and all of the results satisfied the requirements. The method was simple, robust and high‐throughput and is currently being used to provide a TDM service to enhancing therapeutic efficacy and safety use of the PB and PE.