Saponins are natural surfactant compounds whose aqueous solutions can form stable foam and micellar solutions. Gypsophila arrostii Guss., Caryophyllaceae, represents the main source of saponins for industrial applications, such as vaccine adjuvants. Due to the complex and amphiphilic nature of triterpene saponins, these compounds require sophisticated techniques for their isolation and characterization. The hydromethanolic saponin fractions from the roots of the analyzed plant material were investigated by electrospray ionization ion trap multiple-stage mass spectrometry (ESI-IT-MS) in negative mode. According to the m/z values for the deprotonated molecule [M-H](-) and the characteristic product ions, four known Gypsophila-type saponins were successfully characterized. In addition, four undescribed saponin compounds were detected by the registered ions [M-H](-).