Abstract: Objecitve To detect unknown impurities in raw drug material of cefotiam hexetil. Methods High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was employed for the determination of impurities in cefotiam hexetil. Agilent SB-C₁₈ column (150 mm×2.1 mm i.d., 3.5 μm particles) was used for chromatographic separations of cofotiam hexetil dissolved in deionized water, with mobile phase consisting of (A) 0.1% formic acid and (B) acetonitrile and timed gradient program T (min)/B (%):0/3, 5/3, 15/20, 20/40, 30/60, 40/80. The flow rate was set at 0.3 mL/min with DAD detector wavelength fixed at 254 nm. Electrospray ionization source was applied and operated in positive ion MRM mode. The source voltage was kept at 4 kV and cone voltage was 100 V with the mass range m/z 50-1000. Nitrogen was used as nebulizing gas and the nebulizer pressure was 40 psi. The drying gas temperature was 350℃ and the drying gas flow was 10 L/min. Results Unknown impurities of cefotiam hexetil were identified. Substance 1 was Δ³-isomer of cefotiam hexetil. The structures of 3 other substances were also determined. Conclusion The method is sensitive, rapid and credible for the analysis of cefotiam hexetil and its related impurities, which can be applied in quality control of cefotiam hexetil.