Abstract <p>This study presents a new highly sensitive approach to the determination of eight steroidal sapogenins—hecogenin, neoruscogenin, ruscogenin, gitogenin, diosgenin, sarsapogenin, yamogenin, and tigogenin—by comprehensive 2D high-performance liquid chromatography coupled with tandem mass spectrometry in the multiple reaction monitoring mode. The ionization characteristics of sapogenins were studied under atmospheric pressure chemical ionization with positive ion detection. Tandem mass spectra of the selected precursor ions were analyzed, and the main pathways of their collision-activated dissociation were identified. Combining 2D chromatographic separation with mass-spectrometric detection enabled the development of a method for the simultaneous quantification of steroidal sapogenins, achieving limits of detection from 0.7 to 2.9 μg/L. The approach was validated and successfully applied to the analysis of plant extracts rich in steroidal saponins, including <i>Yucca gloriosa</i>, <i>Yucca treculeana</i>, <i>Yucca filamentosa</i>, and <i>Yucca aloifolia</i>.</p>