A high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of five flavonoids, taxifolin, neoastilbin, astilbin, neoisoastilbin and isoastilbin, contained in rhizoma smilacis glabrae. The optimal conditions of separation and detection were achieved on a Lichrospher C18 column (250 mm x 4.6 mm, 5 microm), with a gradient elution program, detected at 291 nm. The correlation coefficients of all the calibration curves showed good linearity (r>0.999) within test ranges. The relative deviation of this method was less than 3% for intra- and inter-day assays, and the average recoveries (n=3) were between 96.2 and 103.1%. The extraction process was also optimized as 2 h immersion and 30 min sonication in 60% ethanol. Eight samples of rhizoma smilacis glabrae from different locations in China were analyzed. The results indicate that the assay is reproducible and precise and could be readily utilized for the quality control of rhizoma smilacis glabrae.