The EtOAc extract of the roots of Glycyrrhiza uralensis exhibited alpha-glucosidase inhibitory activity. Bioassay-guided fractionation resulted in the isolation of an active prenylflavonoid, glycyrrhisoflavone. Its structure was elucidated by NMR and MS analyses. A simple method to prepare glycyrrhisoflavone from the 95% EtOH extract of the roots of G. uralensis was developed by combination of Diaion HP-20 column chromatography (CC), silica gel CC, and preparative HPLC. An HPLC-PDA method was developed for quantitative determination of glycyrrhisoflavone in the roots of G. uralensis. The sample was extracted with MeOH and analyzed using a reversed-phase column with isocratic elution with CH3CN-H2O (0.06% trifluoroacetic acid) (42:58) at a flow rate of 1.2 mL/min, a column temperature of 40 degrees C, and a detection wavelength of 260 nm. The method allowed the determination of glycyrrhisoflavone in the concentration range of 5-500 microg/mL. The relative standard deviation values of the precision and repeatability were 0.3% and 2.0%, respectively. The limits of detection and quantification were 0.5 microg/mL and 5 microg/mL, respectively. The relative recovery rate was 100.2 +/- 1.8%. Based on the validation results, the HPLC determination method was found to be precise, accurate, and time conservative. This method was applied successfully to nine different root samples of G. uralensis. The amounts of glycyrrhisoflavone in these samples were 15-93 mg/100 g of dried powdered plant material.