Abstract: The glucuronide conjugates of icaritin in rat plasma, urine and feces were analyzed by high performance liquid chromatographytandem mass spectrometry (HPLC-MS/MS). Samples were extracted using C₁₈ solid-phase extraction cartridges, and then separated on a Dikma C₁₈ column. The mobile phase was consisted of V(acetonitrile)∶V(water)∶V (acetic acid)= 45∶55∶1.5. Electrospray ionization (ESI) source was applied. Full scan and second stage mass scan were performed in the negative ion mode. According to the chromatography and MS data, a di-glucuronide conjugate (M1) and two mon-glucuronide conjugates (M2 and M3) of icaritin are identified in rat. M1, M2 and M3 can be detected in plasma and urine, while M2 and M3 can be found in feces after oral administration of icaritin to rat.