A sensitive and selective high performance liquid chromatographic method coupled with DAD detection is presented for quality control of Radix Ilicis Pubescentis. By means of this analytical procedure the major individual constituents (ilexoside O, ilexgenin A, ilexsaponin A1, ilexsaponin B1, liriodendin and acanthoside B) could be quantified simultaneously. LC-ESI-MS was applied for identification of the six compounds in the plant by comparing their m/z value and retention times with those of selected standards. For quantitative analysis, the extraction procedure and the extraction solvent were optimized in order to ensure the exhaustive extraction of the plant material. The HPLC conditions were evaluated and optimized for the exact quantification of all six individual compounds. Chromatographic separation was carried out on a C18 column using gradient elution with acetonitrile and 0.1% phosphoric acid as the mobile phase. Detection was carried out using a photodiode array detector. The calibration curves for determination of the six constituents showed good linearity over the investigated ranges (r2>0.999). Measurement of intra-day and inter-day variability (expressed as RSD value) was conducted to assess precisions of the method, and RSD (%) of intra- and inter-day variation were between 1.56-3.36% and 1.61-3.58%, respectively. The recoveries of the six compounds were between 96.4-102.2%, with RSD (%) values ranging from 1.7-3.8%. These validation results demonstrated the suitability of the method for the precise and accurate determination of the main constituents in Radix Ilicis Pubescentis. The method was successfully applied for quality evaluation of 12 batches of Radix Ilicis Pubescentis obtained from different regions of southern China. The contents of the six major constituents varied significantly due to their different origins, which can be used as an aid to assessing the quality of Radix Ilicis Pubescentis.