The application of liquid chromatography-ion trap mass spectrometry for the characterization of linear and cyclic polypeptide antibiotics was investigated. The aim was on-line identification of impurities in those antibiotic complexes without recourse to time-consuming isolation and purification procedures. Hyphenated techniques, such as liquid chromatography coupled to mass spectrometry, are ideally suited for this purpose. Characterization was performed with an ion trap mass spectrometer offering MS(n) capability; this enables more structural information to be obtained. Liquid chromatography in combination with ion trap mass spectrometry was successfully applied for the characterization of impurities in gramicidin, polymyxin B, polymyxin E, and bacitracin and the study of the degradation products of polymyxins B and E.