Thirteen unknown impurities and isomers in cefminox sodium were separated and characterized by liquid chromatography coupled with high-resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF-MS) with the positive mode of electrospray ionization (ESI) method. New HPLC-gradient elution method was developed for the detection of impurities in cefminox sodium. And the ESI ion trap multiple-stage tandem mass spectrometry had been applied successfully to the direct investigation of impurities and isomers in cefminox sodium. The fragmentation patterns and structural assignment of these impurities were studied. Full scan liquid chromatography-mass spectrometry (LC-MS) was first performed to obtain the m/z value of the protonated molecules and formulas of all detected peaks, LC-MSn (n = 1-6) were then carried out on the compounds of interest. Structures of 13 degradation products in cefminox sodium were deduced based on the high-resolution MSn (n = 1-6) data, assisted by the UV spectra and stress testing. And the forming mechanisms of degradation products in cefminox were also studied. The method of LC-IT-TOF-MSn (n = 1-6) was worthy of widespread use and application for the further improvement of official monographs in pharmacopoeias with the advantages of stability and repeatability.