Abstract: A specific ion-pairing HPLC method was developed for the determination of related substances in atropine sulfate. Their structures were identified by LC-MS/MS. HPLC separation was carried out on a Thermo BDS Hypersil C₁₈ column with linear gradient elution by a mobile phase consisting of acetonitrile and phosphate buffer solution(containing sodium heptane-sulphonate, adjusting pH to 5. 0 with phosphoric acid). The flow rate was 0. 8 mL/min and UV detection wavelength was 225 nm. Atropine sulfate and its related substances can be completely separated under the established HPLC condition. And they were characterized by hyphenated chromatographic techniques with accurate mass determination using high resolution LC-TOF/MS methods as well as product MS spectra scanning through a volatile mobile phase. Twelve related substances were detected and identified as by-products from synthesis and degradations of atropine sulfate. Six of them were related substances from EP and the other six were unknown related substances. This newly established HPLC method is effective in the separation and identification of the related substances in atropine sulfate and the results are useful for its quality control and process optimization.