Mahonia leschenaultia(ML) and Mahonia napaulensis(MN) are less known and unexplored medicinal plants of the family Berberidaceae. They are used by the Todas of Nilgiris in their religious and medical practices but chemically less identified. Hence, we decided to do extensive phytochemical analysis to explore the potential of these plant extracts. An ultrahigh performance electrospray tandem mass spectrometry(UHPLC–ESI–MS/MS)method was successfully developed for qualitative analysis of the bioactive components in Mahonia species using Orbitrap Velos Pro mass spectrometer. Sixteen compounds were identified by comparison of their retention times and mass spectra(MS) with authentic standards and reported literature. Multi-stage mass spectra(MS~(2–8)) for the identification of protoberberine and aporphine alkaloids showed the sequential expulsion of all the substituents attached with their basic skeleton followed by CO loss. Eight of the identified compounds(berberine, jatrorrhizine, palmatine, magnoflorine, isocorydine, glaucine, tetrahydropalmatine and tetrahydroberberine) were simultaneously determined by another UHPLC–ESI–MS/MS method under the multiple reactions monitoring(MRM) mode quantitatively using triple quadrupole linear ion trap mass spectrometer. The analytical method was validated for 8 bioactive compounds with overall recovery in the range98.5%–103.6%(RSD≤2.2%), precise(RSD≤2.07%) and linear(r≥0.9995) over the concentration range of 0.5–1000 ng/mL and successfully applied in ML and MN roots, which suggests the suitability of the proposed approach for the routine analysis of Mahonia species and their quality control. Mahonia leschenaultia (ML) and Mahonia napaulensis (MN) are less known and unexplored medicinal plants of the family Berberidaceae. They are used by the Todas of Nilgiris in their religious and medical practices but chemically less identified. Therefore, we decided to do extensive phytochemical analysis to explore the potential of these plant extracts. An ultrahigh performance electrospray tandem mass spectrometry (UHPLC-ESI-MS / MS) method was successfully developed for qualitative analysis of the bioactive components in Mahonia species using Orbitrap Velos Pro mass spectrometer. Sixteen compounds were identified by comparison of their retention times and mass spectra (MS) with authentic standards and reported literature. Multi-stage mass spectra (MS ~ (2-8)) for the identification of protoberberine and aporphine alkaloids showed the sequential expulsion of all the substituents attached with their basic skeleton followed by CO loss. Eight of the identified compounds (berberine, jatrorrhizine, palmatine, magnoflorine, isocorydine, glaucine, tetrahydropalmatine and tetrahydroberberine) successively by another UHPLC-ESI-MS / MS method under the multiple reactions monitoring (MRM) mode quantitatively using triple quadrupole linear ion trap mass spectrometer. The analytical method was validated for ML), linear (r ≧ 0.9999) over the concentration range of 0.5-1000 ng / mL and successfully applied (RSD ≤ 2.2%) in ML and MN roots, which suggests the suitability of the proposed approach for the routine analysis of Mahonia species and their quality control.