Abstract: To identify the related substances of cyclosporin A by LC-MS techniques, the separation of cyclosporin A and its related substances was carried out on a Hypersil BDS C₁₈(100 mm×4. 6 mm, 2. 4 μm)column with isocratic elution by a mixture of acetonitrile-water-MTBE and formic acid(430 ∶520 ∶50 ∶1)as the mobile phase. Cyclosporin A and its 29 related substances(9 process related and 20 degradants)were well separated under the established conditions. Among them, 13 were listed in EP and the rest 16 were unknown products having not been reported before. Electrospray positive ionization high resolution TOF/MS was used for the determination of the accurate mass and elemental composition of parent ions of all the components, and triple quadrupoles tandem mass was employed for the product mass spectra determination. Thence, the structures of all the 29 detected substances were successfully characterized through spectra elucidation and the fragmentation pathways analysis. The established LC-MS method was successfully employed for the separation and identification of the related substances of cyclosporin A and it is useful for its fermentation processes and quality control.