Asterothamnus centrali-asiaticus, a kind of characteristic shrub abundant in grassland and desert areas, has been used as forage fodder for camels and goats in Central Asia, and this plant also plays a critical role in the maintenance of desert grassland ecosystems as a result of its tolerance to poor soils and sand burial. However, its chemical composition has been rarely reported. In this study, phytochemical investigation of this pasturage was performed and three new triterpenoid saponins (1–3) were isolated together with nine known triterpenoid saponins (4–12) using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography (2D RPLC/HILIC). Their structures were elucidated via diverse spectroscopic analyses, including infrared (IR) spectrometry, high-resolution electrospray ionization mass spectrometry (HR-ESIMS), and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR). All isolated triterpenoid saponins (1–12) were reported from this genus for the first time, and they were further evaluated for their cytotoxicity against four cancer cell lines (A549, HepG2, MGC-803, and MFC), which indicated that compound 11 showed potent cytotoxicity against the HepG2 cell line, with an IC₅₀ value of 6.85 μg/mL.