A sensitive and rapid high-performance liquid chromatography (HPLC) method with solid-phase extraction (SPE) to simultaneously determine albiflorin and paeoniflorin in rat serum was described. Serum samples were pretreated with solid-phase extraction using Extract-CleanTM cartridges, and the extracts were analyzed by HPLC on a reversed-phase C18 column and a mobile phase of acetonitrile-0.03% formic acid (17:83 (v/v)) with ultraviolet detection at 230nm. Pentoxifylline was used as the internal standard (IS). The linear ranges of the calibration curves were 29-1450ng/ml for albiflorin and 10-2000ng/ml for paeoniflorin. The intra-and inter-day precisions (R.S.D.) were≤10.49% for albiflorin and≤11.29% for paeoniflorin, respectively. Mean recovery was determined to be 89.75% for albiflorin and 85.82% for paeoniflorin. The limit of quantification was 29ng/ml for albiflorin and 10ng/ml for paeoniflorin, respectively. The validated method was applicable to pharmacokinetic studies of albiflorin and paeoniflorin from rat serum after oral administration of Si-Wu decoction. The pharmacokinetic study indicated that albiflorin and paeoniflorin had poor absorption and rapid elimination. This assay result was necessary for the pharmacokinetic evaluation of Si-Wu decoction.