The root of Scutellaria baicalensis, called Huangqin in Chinese, is one of the most commonly used traditional Chinese medicines for the treatment of hepatitis, tumors, diarrhea, and inflammatory diseases. The major chemical constituents of Huangqin are flavonoids. In the present paper, HPLC-DAD-ESI-MS(n) was used to analyze flavonoids in the roots of S. baicalensis. A total of 26 flavonoids were identified or tentatively characterized, including 5 C-glycosides, 12 O-glycosides, and 9 free aglycones. Two C-glycosides, apigenin-6-C-glucyl-8-C-arabinoside and chrysin-6,8-di-C-glucoside, together with some O-glycosides, are reported from S. baicalensis for the first time. This method is simple, reliable and sensitive, and could be used for the quality control of Huangqin and its related preparations.