Objective: To develop an HPLC method for simultaneous quantification of paeonol and rhein in the tissue of rats. Method: The adopted chromatographic column was Phenomsil BDS C18(4.6 mm×250 mm

5 μm)

the mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution (37: 63)

the flow rate was 1.0 mL·min-1

and detection wavelength was set at 274 nm. Methyl alcohol was used to precipitate the protein of the plasma samples and the aurantio-obtusin was used as an internal standard. Result: The linear range of paeonol was Y=-0.055+0.03X(r=0.993 2). The intra-day RSD and inter-day RSD were less than 4.1%. For rhein

the linear range was Y=-0.152+0.014X(r=0.993 5)

the intra-day RSD and inter-day RSD were less than 4.9%.The stability of the plasma sample were well acceptable. Conclusion: The developed method for the quantification of paeonol and rhein in rat plasma is suitable for the distribution studies of drug-couple Radix Paeoniae Rubra and Rhei Radix et Rhizoma in the tissue of rats.