An investigation was carried out for the impact of two pretreatment methods on the matrix effect of Epimedium-derived flavonoid compounds in rabbit serum by liquid chromatography-tandem mass spectrometry（LC-MS/MS）.Before and after the pretreatment of rabbit blank serum via the extractions of ethyl acetate（liquid-liquid extraction,LLE）and C18 cartridge（solid phase extraction,SPE） respectively,a certain concentration solution of mixed reference substances containing four Epimedium-derived flavonoid compounds（icariin,icariside Ⅰ,icariside Ⅱ and iicaritin） was added exactly,and LC-MS/MS was adopted for sample analysis.Then,the absolute matrix effects,the recoveries and the process efficiencies of Epimedium-derived flavonoid compounds in rabbit serum were evaluated.The result showed that,after the sample pretreatment of LLE with ethyl acetate,the absolute matrix effects of Epimedium derived flavonoid compounds in rabbit serum were more than 100%,the recoveries and the process efficiencies were much higher and stable,while after the sample pretreatment of SPE with C18 cartridge,the absolute matrix effects of Epimedium derived flavonoid compounds in rabbit serum were less than 100%,the recoveries and the process efficiencies were much lower and unstable.The above results indicated that ionization enhancement can be realized for LC-MS/MS analysis of Epimedium-derived flavonoid compounds in rabbit serum while the recoveriesand the process efficiencies of Epimedium-derived flavonoid compounds were much higher and more stable when using ethyl acetate for sample pretreatment,and this pretreatment method was also suitable for the pharmacokinetic study of Epimedium-derived flavonoid glycosides in rabbits as the serum sample pretreatment.