Abstract: This study established a HPLC-fluorescence detection(HPLC-FLD) assay for determining of rhein in human plasma,urine and feces on a reversed-phase C₁₈ column with a mobile phase of methanol-acetonitrile-1% acetic acid (61 ∶15 ∶24).HPLC-FLD was performed at excitation wavelength 440 nm and emission wavelength 520 nm for rhein and the internal standard.Calibration curves were linear over the ranges of 0.05-10.00 μg/mL in plasma,0.10-25.00 μg/mL in urine and 0.20-100.00 μg/mL in feces.The lowest limit of quantification for rhein was 0.05 μg/mL in plasma,0.10 μg/mL in urine and 0.20 μg/mL in feces.The recovery of this method was greater than 80%.The main pharmacokinetic parameters c_max,t_max,AUC_0-∞,t_1/2 and MRT were (16.0±3.4) μg/mL,(4.0±0.6) h,(152.7±45.7) μg ·h/mL,(5.5±1.1) h and (8.9±2.2) h,respectively.The accumulative excretion rate of rhein in urine and feces were (8.19±1.84)% and 11.00%,separately.This assay was proved to be convenient,accurate,sensitive and suitable for mass determination of biological samples.