A rapid and simple method was developed and validated for the determination of colistin A and B in Mueller-Hinton broth using LC-tandem MS. Both analyte and internal standard (IS) (polymyxin B1) were determined using ESI. The MS data were obtained via the selected reaction monitoring in positive-ion mode. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 0.0241-2.41 μg ml(-1) for colistin A and 0.0439-4.39 μg ml(-1) for colistin B. No interference peaks were found in the blank Mueller-Hinton broth tested. Inter- and intraday precision and accuracy were within 85-115% (coefficient of variation). Colistin was stable in the autosampler for at least 24 h at 4 °C and in Mueller-Hinton broth for at least 120 h at 35 °C. This assay has been successfully used to determine colistin A and B in Mueller-Hinton broth for in vitro pharmacodynamic model studies. Accurate determination of colistin in bacterial growth medium has a vital role in the studies examining dosage regimen of and bacterial resistance to colistin.