Picrorhiza kurroa, an "Indian gentian," a known Himalayan medicinal herb with rich source of phytochemicals like picrosides I, II, and other glycosides, has been traditionally used for the treatment of liver and respiratory ailments. Picrosides anti-proliferative, anti-oxidant, anti-inflammatory and other pharmacological properties were evaluated in treating triple-negative breast cancer (TNBC). Picroside I and II were procured from Sigma-Aldrich and were analyzed for anti-cancer activity in triple-negative breast cancer (MDA-MB-231) cells. Cell viability was analyzed using MTT and trypan blue assays. Apoptosis was analyzed through DNA fragmentation and Annexin V/PI flow cytometric analysis. Wound healing and cell survival assays were employed to determine the inhibition of invasion capacity and anti-proliferative activity of picrosides in MDA-MB-231 cells. Measurement of intracellular ROS was studied through mitochondrial membrane potential assessment using DiOC6 staining for anti-oxidant activity of picrosides in MDA-MB-231 cells. Both Picroside I and II have shown decreased cell viability of MDA-MB-231 cells with increasing concentrations. IC₅₀ values of 95.3 µM and 130.8 µM have been obtained for Picroside I and II in MDA-MB-231 cells. Early apoptotic phase have shown an increase of 20% (p < 0.05) with increasing concentrations (0, 50, 75, and 100 µM) of Picroside I and 15% (p < 0.05) increase with Picroside II. Decrease in mitochondrial membrane potential of 2-2.5-fold (p < 0.05) was observed which indicated decreased reactive oxygen species (ROS) generation with increasing concentrations of Picroside I and II. An increasing percentage of 70-80% (p < 0.05) cell population was arrested in G0/G1 phase of cell cycle after Picroside I and II treatment in cancer cells. Our results suggest that Picroside I and II possess significant anti-proliferative and anti-cancer activity which is mediated by inhibition of cell growth, decreased mitochondrial membrane potential, DNA damage, apoptosis, and cell cycle arrest. Therefore, Picroside I and II can be developed as a potential anti-cancer drug of future and further mechanistic studies are underway to identify the mechanism of anti-cancer potential.