Thirteen unknown impurities in flomoxef sodium were separated and characterized by liquid chromatography coupled with high resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF MS)with positive and negative modes of electrospray ionization method for further improvement of official monographs in pharmacopoeias. The fragmentation patterns of impurities in flomoxef in the negative ion mode were studied in detail, and new negative-ion fragmentation regularities were discovered. Chromatographic separation was performed on a Kromasil C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of (A) ammonium formate aqueous solution (10 mM)–methanol (84:16, v/v) and (B) ammonium formate aqueous solution (10 mM)–methanol (47:53, v/v). In order to determine the m/z values of the molecular ions and formulas of all detected impurities, full scan LC–MS in both positive and negative ion modes was firstly executed to obtain the m/z value of the molecules. Then LC–MS2 and LC–MS3 were carried out on target compounds to obtain as much structural information as possible. Complete fragmentation patterns of impurities were studied and used to obtain information about the structures of these impurities. Structures of thirteen unknown degradation products in flomoxef sodium were deduced based on the high resolution MSn data with both positive and negative modes. The forming mechanisms of degradation products in flomoxef sodium were also studied.