Liquid chromatography (LC) methods compatible with mass spectrometry (MS) that are suitable for impurity profiling of vancomycin mixtures have not been described in the literature. The mobile phases of the existing methods contain non-volatile additives and/or solvents that give problems in combination with MS. In this paper, a reversed-phase LC/tandem mass spectrometry method is described for the investigation of vancomycin and related substances. The LC method uses a Zorbax Extend C18 column (250 × 4.6 mm i.d.), 5 µm, and a mobile phase consisting of methanol, water and ammonium acetate solution (pH 9.0). This method allows us to separate vancomycin and its impurities. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray interface operated in the positive and negative ion modes. The LCQ is ideally suited for identification of impurities and related substances because it provides on-line LC/MSn capability, which allows efficient identification without time-consuming isolation and purification procedures. Using this method, the fragmentation of vancomycin and known derivatives was studied and the structures of six substances occurring in commercial samples were elucidated. Copyright © 2006 John Wiley & Sons, Ltd.