Abstract: Hyphenated methods of chromatography and high resolution mass spectrometry were established for the identification of the related substances in cisatracurium besylate for injection.The analysis was conducted on SEPAX GP-C₁₈ column by isocratic elution using a mixture of water,acetonitrile,methanol,formic acid and ammonium formate in the ratio of 650 ∶175 ∶175 ∶10 ∶1.3 as the mobile phase at the flow rate of 1.0 mL/min.The related substances were detected and identified by LC/TOF-MS and LC-MS/MS with acetonitrile aided electrospray ionization.Eleven related substances were found in cisatracurium besylate for injection,seven of which had the area normalization content of over 0.1%.Based on the structural characterization of the text sample，the formula were speculated through the exact mass obtained by LC/ESI-TOF-MS using Mass Hunter software.All the related substances were preliminarily elucidated using the spectra as well as the organic reaction mechanisms,five of which were not listed in the corresponding monographs of official pharmacopoeias.