Abstract: AIM To analyze the related substances in ampicillin and amoxicillin and determine the related substances in products. METHODS By using HPLC, electrospray ionzation and selective ion minitoring mass spectrum method, accelerated samples were analyzed for determination the related substances in ampicillin and amoxicillin. RESULTS Thirteen related substances in ampicillin and 9 related substances in amoxicillin were determined. CONCLUSION The results are useful to quality control and stability study for ampicillin and amoxicillin.