The quality control of natural products is principal key to guarantee the Good Manufacturing Practices (GMP) and Good Clinical Practices (GCP) for the functional food, pharmaceuticals and cosmeceuticals in the industry. In this study, we examined the quantitative analysis of berberine as marker substance of Coptidis Rhizoma by high performance liquid chromatography-photodiode array detector (HPLC-DAD). The HPLC method was validated and met all the requirements for the quality control analysis recommended by FDA and ICH. The berberine was separated on a Xterra C18 column (5 ㎛, 4.6 × 250 ㎜) using mobile phase consisting of distilled water and acetonitrile with KH2PO4 (3.4 g) and Na2SO4 (1.7 g). Calibration curve of berberine has been estimated (y = 42293.47x-41589 with the correlation coefficient 0.9999). The amount of berberine was calculated as 4.25%. And berberastine, palmatine, columbamine, jatrorrhizine, epiberberine, berberine and coptisine in the Coptidis Rhizoma were identified by high performance liquid chromatography - electrospray ionization-mass spectrometer (HPLC-ESI-MS) method.