Objective:To study the varieties and contents of related substances in cefalotin sodium by HPLC and MS. Methods:The quantitative determination of impurity spectra was carried out using self-contrast method, and UPLC-MS was used to preliminarily i-dentify the variety of impurities. Results:Through the study on the ascription and content of the related substances, there were 6 sub-stances existing in cefalotin sodium, and through the structure analysis, the 6 substances showed antibiotic action and toxicity. Conclu-sion:The method is simple, stable and repeatable, which can be used for the determination of cefalothin sodium and related sub-stances.