The European Pharmacopoeia (Ph. Eur.) prescribes a selective and sensitive liquid chromatography/ultraviolet (LC-UV) method for the separation of the 16-membered ring macrolide josamycin and its related compounds. Since josamycin is obtained by fermentation, several closely related substances can be found in the sample. Several impurities have already been identified using reference substances. However, many peaks in the chromatogram cannot be correlated with known compounds or correspond to structures which were not described previously. The hyphenation of LC to mass spectrometry (MS) is a very useful tool for the characterization of impurities. The existing LC-UV method however uses non-volatile buffers, while for LC/MS a volatile mobile phase is required. In this study, each peak from the non-volatile system was collected separately and reinjected into a LC system using volatile mobile phase constituents. This way, the analyte could be separated from the buffer salts. Mass spectral data of this macrolide antibiotic were acquired on a LCQ ion trap mass spectrometer, equipped with an electrospray ionization (ESI) probe operating in the positive ion mode. The identity of the unknown compounds was deduced using the MS/MS and MS(n) collision-induced dissociation spectra of reference substances, combined with knowledge about the nature of functional group fragmentation behavior. The impurity profiling was done on 30 peaks in a josamycin bulk sample. This way, 12 compounds reported in the literature and Ph. Eur. were found in the bulk sample. Furthermore, 12 novel related substances were characterized and 18 compounds were partially characterized.