Objective:To develop an HPLC method for simultaneous determination of epimedin C and icariin in 21 species of Epimedium.Methods:The samples were separated on Elite SinoChrom ODS-AP column(250 mm×4.6 mm,5 μm)by gradient elution with acetonitrile(A)-water(B)(0-8 min,27%A;8-30 min,27%A→29%A)as the mobile phase.The flow rate was 1 mL·min-1,and the detection wavelength was 270 nm.Results: The linear ranges of epimedin C and icariin were 0.130-3.89 μg(r=0.9999),0.0294-1.47 μg(r=0.9999)respectively;The average recoveries(n=9)of epimedin C and icariin were 103.9%,100.0% respectively;The contents of epimedin C and icariin were 0.02%-7.80%,0.01%-1.74% respectively.Conclusion:This method is simple,quick,sensitive,reliable and reproducible.It is the best choice for the determination of epimedin C and icariin in Epimedium.