A new HPLC method for the determination of paeoniflorin in rat serum with solid-phase extraction (SPE) for preconcentration is introduced. Paeoniflorin and an internal standard (pentoxifylline) were extracted from serum by means of SPE using cartridges with octadecyl chemically bound phase. The HPLC separation was then performed on a reversed-phase C18 column using acetonitrile-water (18:82, v/v) as eluting solvent system, and UV detection at 230nm to measure the analyte with a limit of quantitation about 10ngml-1. The calibration curve for paeoniflorin was linear (r=0.9938) in the concentration range of 10-1200ngml-1, both intra-and inter-day precision of the paeoniflorin were determined and their coefficience of variation did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of paeoniflorin from rat serum after oral administration of Guan-Xin-Er-Hao decoction.