Abstract A method for quantitative determination of oridonin in rat plasma using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with UV spectrometry was established and the method was applied to a pharmacokinetics study of oridonin in rats. From a variety of compounds and solvents tested, ticolpidine was selected as the internal standard (IS) and ethyl acetate was found to be the best solvent for extracting oridonin from plasma samples. RP-HPLC analysis of the extracts was performed on an analytical column (DIKMA ODS, 200 mm × 4.6 mm; i.d., 5 μm) equipped with a security guard pre-column system. There was a good linearity over the range 0.05–8.0 μg/mL ( r > 0.99). The recoveries were about 95.0% in plasma, and the intra- and inter-day coefficients of variation were less than 9.0% in all cases. The limit of detection (LOD) was 0.025 μg/mL and the lower limit of quantification (LLOQ) was 0.05 μg/mL. The RP-HPLC method was readily applied to quantitate oridonin in rat plasma within 24 h in a pharmacokinetics study where experimental rats received a single dose of oridonin (12.5 mg/kg) and the result was presented.