A LC-MS method for identification of the components and related substances in gentamicin sulfate was developed by optimizing preparations of samples, mobile phase and chromatog. columns.Based on MS spectrometric data and references, the structures of the related substances of gentamicin sulfate were elucidated.The unknown related substances detected in the HPLC with integrated pulsed amperometry detector (IPAD) were identified.The results indicated that there were some impurities, such as Garamine, Gentamicin A3, Gentamicin X2, Gentamicin B, Gentamine C2, JI-20 A, VII-3, Gentamine C2 a, Gentamicin B1 and Sisomicin in Gentamicin sulfate product.The scientific information could be used for production process improvement and quality control.