Rhein (4,5-dihydroxy-9,10-dioxoanthracene-2-carboxylic acid, cassic acid) is a pharmacological active component found in Rheum palmatum L. the major herb of San-Huang-Xie-Xin-Tang (SHXXT), a medicinal herbal product used as a remedy for constipation. Here we have determined multiple bioactive components in SHXXT and investigated the comparative pharmacokinetics of rhein in rats. A sensitive and specific method combining liquid chromatography with electrospray ionization tandem mass spectrometry has been developed and validated to simultaneously quantify six active compounds in the pharmaceutical herbal product SHXXT to further study their pharmacokinetics in rats. Multiple reaction monitoring (MRM) was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. There were no significant matrix effects in the quantitative analysis and the mean recovery for rhein in rat plasma was 91.6%±3.4%. The pharmacokinetic data of rhein demonstrate that the herbal formulae or the single herbal extract provide significantly higher absorption rate than the pure compound. This phenomenon suggests that the other herbal ingredients of SHXXT and rhubarb extract significantly enhance the absorption of rhein in rats. In conclusion, the herbal formulae (SHXXT) are more efficient than the single herb (rhubarb) or the pure compound (rhein) in rhein absorption.