Abstract: To identify the related substances of fusidic acid by LC-MS, separation was performed on an Agilent Extend-C₁₈ column(150 mm × 4. 6 mm, 3. 5 μm)by linear gradient elution with a mobile phase consisting of methanol, acetonitrile and formic acid. Electrospray positive ionization high resolution TOF/MS was used for the determination of the accurate mass and elemental composition of parent ions of the related substances; triple quadrupole tandem mass was employed for the mass spectra determination of the product. The structures of the related substances were then figured out through the elucidation of the fragment ions. Fusidic acid and its related substances were adequately separated under the established HPLC conditions. Nineteen major related substances of fusidic acid were detected and speculated by hyphenated techniques. Eleven of them were recorded in European Pharmacopoeia, while the others have not been previously reported. The established LC-MS method is effective for the separation and identification of the related substances of fusidic acid and the results are useful for its storage conditions and quality assurance.