Background and objective: Holostemmaadakodien, a plant root extract, has shown potential anticancer activity. The study aims to analyze the anticancer activity of Holostemmaadakodien root extract using acetone extract and to determine the mechanism by which Holostemmaadakodien induces cell death in cancer cells, specifically through apoptosis. Materials and Methods: The breast cancer cells MDA MB 231 and MCF - 7 were used for the cytotoxicity assay. The cells were grown in a specific culture medium with fetal calf serum and antibiotics.H. adakodien Acetone fraction, an annexin staining kit, rabbit polyclonal antibodies, and Cytochrome C were purchased for the experiment.Different staining methods were used to characterize apoptotic cell death, including staining of condensed nuclei and annexinbinding.Cell viability was assessed using the MTT assay and cell counting was performed using trypan blue dye exclusion assay. Results: H-adakodien inhibits the growth of human breast cancer cells MDA MB231 and MCF-7 in a concentration-dependent manner.The degree of cell death induction is found to be dose-dependent, ranging from 37.5 to 300 pg/mL.The acetone extract of H-adakodien shows a clear concentration-dependent inhibition of growth in the breast cancer cells. Treatment with H-adakodien results in morphological alterations, nuclear condensation, and translocation of phosphatidyl serine in the cancer cells. The cytotoxic effects of H-adakodien involve apoptotic changes, as evidenced by nuclear condensation observed in the treated cells.H-adakodien induces apoptosis, not necrosis, in the breast cancer cells. H-adakodien causes nuclear fragmentation in the cancer cells, a characteristic feature of apoptosis. Conclusion: The analysis indicates that the treatment of cancer cells with Holostemmaadakodien root extract leads to cell death through the activation of the apoptosis cascade.