Abstract: An LC-TOF/MS and LC-MS/MS method was established for the identification the related substances in ambrisentan. HPLC separation was carried out on an XBridge C₁₈ column(4. 6 mm×150 mm, 3. 5 μm)with linear gradient elution using a mobile phase consisting of acetonitrile, water and 0. 15% formic acid. The structures of the related substances were identified by electrospray positive ESI high resolution TOF/MS and MS/MS spectra, and verified further through reference substances. Ambrisentan and its related substances can be separated with the established HPLC conditions. Ten related substances were detected and identified. The established method is useful for the identification of related substances in ambrisentan. The results obtained are valuable for its manufacturing process optimization and quality control.