Tulbaghia violacea is a widely known anticancer herb used as traditional medicine in the Southern African region. However, the identities of the chemical compounds responsible for the medicinal properties are still at large. Isolation of the pro-apoptotic compounds from T. violacea was carried out using apoptosis induction-guided fractionation. The chemical structures of the pro-apoptotic molecules were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D nuclear magnetic resonance (NMR), infrared (IR) and elemental analysis. Pro-apoptotic glucopyranosides Dfructofuranose-(26)-methyl--D-glucoyranoside and -D-fructofuranosyl-(26)--D-glucopyranoside were uncovered from aqueous whole plant extracts of T. violacea using bioactivity-guided purification. Glucopyranosides D-fructofuranose-(26)-methyl--D-glucoyranoside and -D-fructofuranosyl-(26)- -D-glucopyranoside have structural similarity with the earlier reported methyl -D-glucopyranoside from the same herb, which selectively kills cancer cells through apoptosis mechanisms. The discovery of these molecules could contribute towards the development of new anticancer drugs.