Objective To development the method of separating and determining the related substances for injection of ampicillin sodium/sulbactam sodium.Methods HPLC gradient elution was performed by using Phenomenex C18(2) colum,named Luna.The mobile phase was consisted of A,0.02 mol/L phosphate buffer(pH4.0±0.1);and B,acetonitrile.The flow rate was 1.0ml/min,the detection wavelength was 230nm/254nm.The concentration of related substances was quantified in different division by double wavelengths.The impurities of sulbactam, before the main peak of sulbactam, was quantified by external standard method of reference sulbactam in 230nm, and the impurities of amp icillin, after the main peak of sulbactam, was quantified by external standard method of reference amp icillin at 254nm. Results　The isocratic elution method was available for detecting amp icillin dimer, (2S)-2-amino-3-methyl-3-sulphinobutanoic acid and other impurities. The impurities from different sources had obvious division, and the impurities of ampicillin mainly the peak of sulbactam, the impurities of sulbactam mainly before this peak. It was able to relate the result of related substances in the injection with the impurities in the single drug of ampicilln or sultactam, that detected in different division by double wavelengths. Conclusion 　The improved method can be used to separate the related substances from different components, and is suitable for controlling the quality of this drug.