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<b>Background/Objectives</b>: Saponins have recently emerged as promising natural products that enhance toxin-based anticancer therapeutics by improving cytosol uptake. This study aimed to identify structurally defined novel natural saponins and evaluate their ability to enhance anticancer cytotoxicity. <b>Methods</b>: The roots of <i>Saponaria officinalis</i> L. were extracted with aqueous ethanol and purified by silica gel column chromatography and reverse-phase high-performance liquid chromatography (RP HPLC). The structures of new saponins were elucidated by NMR spectroscopy and mass spectrometry. Biological activity was assessed in vitro using multiple cancer cell lines. <b>Results</b>: Two pairs of structurally defined pure saponins were obtained: SO1406 and SO1448, and SO1684 and SO1726. Structural elucidation revealed that SO1684 and SO1726 share the core structure 3-<i>O</i>-<i>β</i>-D-Gal-(1→2)-[<i>β</i>-D-Xyl-(1→3)]-<i>β</i>-D-GlcA-gypsogenin-28-<i>O</i>-<i>β</i>-D-Qui-(1→4)-[<i>β</i>-D-Xyl-(1→3)-<i>β</i>-D-Xyl-(1→4)]-<i>α</i>-L-Rha-(1→2)-<i>β</i>-D-Fuc, with SO1684 acetylated at Qui O-4 and SO1726 bearing additional acetylation at Qui O-3. Deacetylation of either SO1684 or SO1726 afforded a known saponin SA1641 isolated from Saponinum album (Merck). Similarly, SO1406 and SO1448 were identified as 3-<i>O</i>-<i>β</i>-D-Gal-(1→2)-[<i>β</i>-D-Xyl-(1→3)]-<i>β</i>-D-GlcA-gypsogenin-28-<i>O</i>-<i>β</i>-D-Xyl-(1→4)-<i>α</i>-L-Rha-(1→2)-<i>β</i>-D-Fuc derivatives, each acetylated at Fuc O-4, with SO1448 containing an additional acetyl group at Fuc O-3. Among the isolated compounds, SO1684 is a known saponin and SO1406 exhibited the most pronounced biological activity, significantly enhancing the cytotoxicity of the ribosome-inactivating protein saporin (SAP) in the MDA-MB231 (triple-negative breast cancer) cell line. <b>Conclusions</b>: SO1406 demonstrates strong cytotoxicity-enhancing activity, highlighting the significant potential of structurally defined natural saponins to advance intracellular delivery and improve the therapeutic performance of protein-based anticancer agents.