A new method for analysis of vitexin and isovitexin in Crotalaria sessiliflora L.was developed by focused microwave-assisted aqueous two-phase extraction(FMAATPE) coupled with high performance liquid chromatography(HPLC).The experimental factors，including ethanol concentration，ratio of material to solvent，microwave power and extraction time were optimized based on the single factor test and orthogonal test.The analysis was performed by HPLC with UV detection at 340 nm.The mobile phase consisted of acetonitrile-water containing 0.5% phosphoric acid(14∶86).The optimum extraction conditions were as follows:two-phase composition:35% ethanol-16% ammonium sulfate，ratio of material to solvent:1∶50，microwave power:140 W， extraction time:20 min.The method was applied in the determination of vitexin and isovitexin in Crotalaria sessiliflora L.，with recoveries of 96.9%-103.8% and RSDs of 1.9%-2.6%.