There is increasing interest in the optimization of polymyxin B dosing regimens to treat infections caused by multidrug-resistant Gram-negative bacteria. We aimed to develop and validate a liquid chromatography-single quadrupole mass spectrometry (LC-MS) method to quantify polymyxin B in two growth media commonly used in in vitro pharmacodynamic studies, cation-adjusted Mueller-Hinton and tryptone soya broth. Samples were pre-treated with sodium hydroxide (1.0. M) and formic acid in acetonitrile (1:100, v/v) before analysis. The summed peak areas of polymyxin B1 and B2 relative to the summed peak areas of colistin A and B (internal standard) were used to quantify polymyxin B. Quality control samples were prepared and analyzed to assess the intra- and inter-day accuracy and precision. The robustness of the assay in the presence of bacteria and commonly co-administered antibiotics (rifampicin, doripenem, imipenem, cefepime and tigecycline) was also examined. Chromatographic separation was achieved with retention times of approximately 9.7. min for polymyxin B2 and 10.4. min for polymyxin B1. Calibration curves were linear between 0.103 and 6.60. mg/L. Accuracy ( relative error) and precision ( coefficient of variation), pooled for all assay days and matrices (n= 84), were -6.85 (8.17 ) at 0.248. mg/L, 1.73 (6.15 ) at 2.48. mg/L and 1.54 (5.49 ) at 4.95. mg/L, and within acceptable ranges at all concentrations examined. Further, the presence of high bacterial concentrations or of commonly co-administered antibiotics in the samples did not affect the assay. The accuracy, precision and cost-efficiency of the assay make it ideally suited to quantifying polymyxin B in samples from in vitro pharmacodynamic models.