To develop a HPLC-DAD method for simultaneous determination of six flavonoids in Scutellaria baicalensis, and study the effect of geographic sources, culturation, harvesting time and processing on the contents of flavonoids.The analysis was performed on a Thermo ODS-2 Hypersil column (4.6 mm x 250 mm, 5 microm) at 26 degrees C with a gradient program of acetonitrile-0.1% H3 PO4 aqueous solution-tetrahydrofuran as the mobile phase and a Diode Array Detector as the detector with the detection wavelength at 274 nm. The flow rate was 1.0 mL x min(-1), and the injection value was 20 microL.Six flavonoids, baicalin (1), oroxylin A-7-O-glucuronide (2), wogonoside (3), baicalein (4), wogonin (5) and oroxylin A (6), were linear over the selected range with R2 > or = 0.999 3. The average recoveries were between 96.6% -103.0%, and RSD were less then 5.0% (n = 9) for flavonoids 1-6. By analyzing the data obtained, the effect of geographic sources, culturation, harvesting time and processing on the contents of flavonoids were discussed.This method is simple, sensitive, reliable and reproducible. It can be used for the quality control of S. baicalensis.