Cancer stem cells (CSCs) are characterized by their ability to self-renew, to differentiate into multiple cell types and also drive tumor formation, altogether making them important cellular targets for therapeutic intervention. However, existing CSC-targeting drugs do not significantly improve clinical outcomes. More recently, preclinical studies of natural product-derived compounds have demonstrated their potential usefulness as a therapeutic cancer treatment through their cytotoxic actions on CSCs.Here, we identify CSC-specific compounds derived from natural products and characterize their putative mechanisms of action in CSCs.Glioblastoma stem cells (GSCs) were labeled with EGFP via homologous recombination and utilized for a high-throughput screen of 8,344 fractions from 386 herbal medicines. The fractions that extinguished EGFP fluorescence signal were then further characterized by LC-MS/MS. Next, several putative cytotoxic compounds were evaluated for their cytotoxic effects on GSCs, cancer cell lines and immortalized cells using a variety of methods to study cell proliferation (EdU incorporation assay), cell death (cleaved-Caspase-3 immunostaining), DNA damage (comet assay), mitochondrial membrane changes (JC-1 immunostaining), and tumor formation in vitro (soft agar colony forming assay). We also performed surface plasmon resonance analysis, western blotting, and immunohistochemistry to characterize the putative mechanisms underlying the cytotoxic effects of putative compounds on GSCs. Finally, we carried out xenograft tumor growth assays to study the cytotoxic potential of several candidates in vivo.Our high throughput screen led to the identification of the furostanol saponin taccaoside A and its two homologs from the rhizomatous geophyte Tacca. subflabellata that were cytotoxic to GSCs. Interestingly, the cytotoxic effect of taccaoside A on cell lines was significantly less compared to its homologs, owing to stereochemical differences of a carbon-carbon double bond between C-20 and C-22. Molecular studies revealed that taccaoside A binds to RAS to inhibit downstream effector signaling. Correspondingly, blockade of the interaction between taccaoside A and RAS abolished the inhibitory effect of this compound on CSCs. Furthermore, taccaoside A treatment was effective in limiting tumor cell growth in vivo.Our study yielded an effective approach to screen for CSC-specific agents. Through this approach, we identified taccaoside A from the rhizomatous geophyte Tacca. subflabellata are cytotoxic to CSCs through a molecular mechanism that involves RAS binding and suppression of its downstream signaling. Our findings indicate taccaoside A is a potential lead compound for anti-CSC drug discovery.Copyright © 2022. Published by Elsevier GmbH.