A sensitive, specific and accurate LC-MS/MS method was developed for simultaneous determination of icariin, icariside II and osthole in rat plasma. With carbamazepine as the internal standard, plasma samples were prepared by protein precipitation with acetonitrile. Analysis was carried out on an ACQUITY UPLC BEH C(18) column with a linear gradient and 0.1% aqueous acetic acid and acetonitrile were used as mobile phase. Detection was performed by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring. Linear calibration curves of icariin, icariside II and osthole were obtained over the concentration ranges of 2.00-200, 2.00-200 and 2.00-500 ng/ml, respectively. The intra- and inter-day precisions were within 8.0% and 14%, and the accuracy was from -6.0% to 9.0%. The method was successfully applied to pharmacokinetic studies of icariin, icariside II and osthole in rats after oral administration of Gushudan extract.