Phytochemical investigation from the stem bark of Butea monosperma, led to the isolation and identification of three new compounds named buteaspermin A (1), buteaspermin B (2) and buteaspermanol (3), along with 19 known compounds. The structure of compounds 1-22 were established on the basis of their spectroscopic data. The isolated compounds 2-17 were evaluated using neonatal (1-3 day old) rat calvaria derived primary osteoblast cultures. Five of these compounds 7, 10-13 showed promising osteogenic activity, attributed to increased osteoblast proliferation, differentiation and mineralization as evidenced by marked increase in expression of alkaline phosphatase, an early phase differentiation marker, and alizarin Red S staining of osteoblasts cultured for 48 h and von Kossa silver staining of nodules formed 15 days after culture with these compounds. Quantification of mineralization by optical density measurement of Alizarin Red S extracted from stained osteoblasts cultured for 7 days in presence of these compounds showed significant (P<0.05, vs corresponding vehicle control group) increase in mineralization. On the basis of biological results, structure-activity relationships are discussed.