An ultra performance liquid chromatography coupled with hybrid triple-quadrupole linear ion trap tandem mass spectrometry (UPLC-ESI-QqQLIT-MS-MS) method in multiple reaction monitoring mode was developed for identification and simultaneous determination of potential osteogenic compounds in ethanol extracts of different plant parts of Butea monosperma collected from different geographical regions. The chromatographic separation was carried out on an Acquity UPLC CSH C18 column (1.7 μm, 2.1 × 100 mm) with 0.1% (v/v) formic acid in water and methanol as mobile phase under gradient conditions in 8 min. The developed method was validated according to the guidelines of international conference on harmonization. The correlation coefficients of all the calibration curves were ≥0.9995 and recoveries ranged from 95.2 to 105.8% (RSD ≤ 1.95%). Relative standard deviations of intra-day, inter-day precisions and stability were ≤1.74, 1.84 and 2.8%, respectively. The quantitative results showed remarkable differences in the content of all potential osteogenic compounds in different parts of the plant as well as samples from different geographical regions. Quantitative variations studied from principal component analysis indicated tentative markers for B. monosperma cultivars which can discriminate sample of different geographical regions.