Background: Polygonum multiflorum and Achyranthes bidentate are traditional Chinese herbs, which show significant antiosteoporosis (OP) effect. These two herbs are commonly used as a combination in traditional Chinese medicine. Materials and Methods: In this study, the left humerus section of the mice were stained with hematoxylin and eosin staining to establish the spectrum-effect relationships between the high-performance liquid chromatography fingerprint of different solvent extracts and the anti-OP effect. Finally, we analyzed the correlation between the content of main compounds of different solvent extracts and the anti-OP effect. Results: The compatibility had obvious therapeutic effect on OP induced by retinoic acid. The results of pharmacodynamic analysis showed that the extract had a certain effect on the bone-specific alkaline phosphatase, the level of tartrate-resistant acid phosphatase-5b (TRACP-5b), and estrogen 2 and had a great influence on TRACP-5b especially. The results of spectrum-effect relationships and correlation analysis showed that the level of β-ecdysterone and tetrahydroxystilbene is negatively correlated with bone resorption index and TRACP-5b. The higher the content of the two compounds in the combination, the better the effect of treating mouse OP induced by retinoic acid. Conclusion: It is suggested that the compatibility of P. multiflorum and A. bidentata can be used to treat OP, and their mechanism of action is through reducing osteoclastic effect. β-ecdysterone and tetrahydroxystilbene may be the active components of combination of Polygoni multiflori-A. bidentate.