As an alternative of traditional extraction method, pressurized liquid extraction (PLE) was applied for five flavanoids extraction from Lysimachia clethroide. The operational parameters of PLE, such as extraction solvent, temperature, pressure, static extraction time, flush volume and cycles were optimized by univariate approach coupled with central composite design (CCD) in order to obtain the highest extraction efficiency. The optimized result employed 50% acetonitrile aqueous as extraction solvent, 100 °C of extraction temperature, 1500 psi of extraction pressure, 25 min of static time, 70% flush volume, and only one cycle to extract the target compounds completely. Finally, the contents of five major flavanoids in L. clethroides from different sources were determined simultaneously by the combination of the presented PLE and HPLC method. © 2006.