| Summary: | <i>Yucca schidigera</i> Roezl (Mojave), a kind of ornamental plant belonging to the <i>Yucca</i> genus (Agavaceae), whose extract exhibits important roles in food, beverage, cosmetic and feed additives owing to its rich spirostanol saponins. To provide a comprehensive chemical profiling of the spirostanol saponins in it, this study was performed by using a multi-phase liquid chromatography method combining a reversed phase chromatography T<sub>3</sub> column with a normal phase chromatography silica column for the separation and an ESI-Q-Exactive-Orbitrap MS in positive ion mode as the detector. By comparing the retention time and ion fragments with standards, thirty-one spirostanol saponins were identified. In addition, according to the summary of the chromatographic retention behaviors and the MS/MS cleavage patterns and biosynthetic pathway, another seventy-nine spirostanol saponins were speculatively identified, forty ones of which were potentially new ones. Moreover, ten novel spirostanol saponins (three pairs of (25<i>R/S</i>)-spirostanol saponin isomer mixtures) were targeted for isolation to verify the speculation. Then, the comprehensive chemical profiling of spirostanol saponins from <i>Y. schidigera</i> was reported here firstly.
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