A rapid and simple high-performance liquid chromatographic (HPLC) method was developed and validated to simultaneously analyze the diastereomers of (+)-licarin A and isolicarin A in rat plasma after intravenous administration. The analytes were extracted from the plasma by solid-phase extraction (SPE). Diastereomeric separation and determination were successfully achieved using a Diamonsil ODS C (18) reversed-phase column (250 mm x 4.6 mm i. d., 5 microm) with an RP18 guard column (8 mm x 4.6 mm i. d., 5 microm) and a mobile phase of MeOH-H (2)O (4 : 1, v/v). UV detection was at 270 nm. The linear ranges of the standard curves were 0.25 - 150.00 microg/mL for (+)-licarin A and 0.10 - 75.00 microg/mL for isolicarin A. The lower limits of detection and quantification were 0.05 and 0.25 microg/mL for (+)-licarin A, and 0.05 and 0.10 microg/mL for isolicarin A, respectively. This assay method was successfully applied to study the pharmacokinetics of diastereomers (+)-licarin A and isolicarin A in rat plasma.