Ginsenoside-Rh2 (G-Rh2) is an important constituent in ginseng and has significant anti-tumor property. A simple HPLC assay was developed to study pharmacokinetics of G-Rh2 in rat plasma. The HPLC consisted of a C18 reversed phase analytical column, a variable wavelength ultraviolet (UV) spectrophotometric detector, and a mobile phase made up of 0.005 M KH2PO4 (pH 7.2): acetonitrile: methanol (23:7:70). The system was operated at ambient temperature isocratically at a flow rate of 0.5 mL/min and wavelength at 203 nm. Extraction of G-Rh2 from plasma was achieved by solid phase extraction (SPE) using 100 mg/mL C18 SPE columns. The results showed that standard curves using 50 mL of plasma sample were linear from 0.25 to 100 mg/mL, with regression coefficient (r2)>0.99. The intra- and inter-assay variations over a 3-month study period were <10% and <20%, respectively. After 10 mg/kg twice daily for 4 doses by subcutaneous (sc) injection, the mean maximum plasma concentration (Cmax), and time to Cmax (Tmax) of G-Rh2 was 0.79 mg/mL and <0.5 hour, respectively. The described HPLC is readily performed in most laboratories and should have adequate sensitivity and specificity to study pharmacokinetics of G-Rh2 in rats following multiple doses.