Bone morphogenetic protein II (BMP2), a member of the transforming growth factor-beta (TGF-beta) superfamily, is highly expressed in osteoblasts and is a crucial regulator of osteogenic differentiation. Many observations clearly indicate the high potency of BMP2 as an inducer of osteogenesis, and it may be a novel therapeutic target for diseases associated with bone loss, especially in menopausal and postmenopausal women. To discover new agents that enhance the expression of the mouse BMP2, the authors developed a high-throughput assay to screen a synthetic and natural compound library. The cell-based high-throughput screen was conducted in 96-well plates using the clonal murine calvarial MC3T3-E1 cells. These cells were stably transfected with mouse BMP2 promoter-luciferase and calibrated with the known antiosteoporosis compound genistein. Among 3192 compounds screened, 3 agents (daidzein, formononetin, and 2-Acetyldibenzothiophene) were picked up by the high-throughput screening assay, and those compounds were identified as upregulators of BMP2 expression by real-time quantitative reverse transcription-polymerase chain reaction and flow cytometry. Thus, it is demonstrated that this screening model is useful for identifying lead compounds to treat osteoporosis and maintain bone metabolism balance.