Background: The compatibility of Radix Polygoni multiflori-Achyranthes (RPMA) root is a Traditional Chinese medicine pair frequently used in the clinic and has good anti-osteoporosis (OP) effects. Materials and Methods: The extract of the compatibility of RPMA root was given orally in this study. The pharmacokinetics and the distribution in the tissues of tetrahydroxystilbene glucoside and β-ecdysterone were considered by high-performance liquid chromatography in OP rats induced by retinoic acid and healthy rats. Quercetin was employed as the internal standard. According to the above results, the effects of Osteoporosis on the process of β-ecdysterone and 2,3,5,4'-tetrahydroxy stilbene 2-Ο-β-D-glucoside in rats were analyzed. Results: The results displayed that the peak time of β-ecdysterone in plasma of OP model rats was earlier and the plasma concentration and pharmacokinetic parameters of tetrahydroxystilbene glucoside in the model group were suggestively different from those in the normal group. The concentration distribution of the two components in each tissue of the model group was significantly diverse from that of the normal group. In particular, the concentrations of the two components in kidney tissue of the model group were inferior than those of the normal group at most time points. Conclusion: It is ventured that the two components may be convoluted in the treatment of OP. This experiment can deliver the reference for the further pharmacodynamic study of the compatibility of RPMA root and the correlation analysis of pharmacodynamics and the content of active ingredients in vivo.