Scutellaria baicalensis Georgi is a well-known medicinal plant widely used in China and other East Asian countries. High performance liquid chromatography combined with diode array detection and electrospray ion trap mass spectrometry was used to determine the flavonoid profile of S. baicalensis. Under the optimized experiment conditions, 32 flavonoids were clearly detected. Eighteen main ones were doubtless identified by comparing their retention time, UV and MS (MS(n)) data with isolated or commercial standards. The UV characteristics of these 18 known standards were studied in detail. The rules summarized provided valuable indications for the subsequent on-line identification processes. By interpreting both the MS and the UV data in detail, other 13 minor flavonoids in S. baicalensis were on-line identified successfully.