Abstract: To identify the amino alcohols related substances in atenolol. The related substances in atenolol and its stressed samples were pre-column derivatized with 9-fluorenylmethyl chloroformate. The separation was carried out on an Inertsil ODS-SP column (250 mm×4.6 mm, 5 μm) with linear gradient elution by methanol-ammonium acetate solution as the mobile phases. Electrospray positive ionization high-resolution Q-TOF/MS was used for the determination of the accurate masses and elemental compositions of the parent and fragment ions of these related substance derivatives. The structures of all the detected substances were identified by spectral analysis and synthetic analysis. Under the established conditions, atenolol and its amino alcohols related substances were well separated, and a total of 14 impurity peaks were detected and identified, of which 12 were related substances and 2 were derivatization reaction by-products. The established LC-MS method provides a reference for the examination and quality control of atenolol related substances.