INTRODUCTION: Breast cancer has become the most frequently diagnosed cancer worldwide. Beta-sitosterol and its derivatives have been explored for its anticancer properties. Therefore, this study aims to analyze the testing procedure carried out on MCF7 and MDA-MB-231 breast cancer cells, as well as MCF 10A non-cancerous breast epithelial cells. METHODS: The compounds tested included β-sitosterol and its derivatives: 3β-galactose sitosterol, sitostenone, 3β-glucose sitosterol, poriferasta-5, 22E, 25-trien-3β-ol, and 22-dehydrocholesterol. Cytotoxicity assay was conducted using the PrestoBlue™ Cell Viability Reagent on MCF-7, MDA-MB-231, and MCF 10A cells. The compounds with the highest and lowest cytotoxicity were further analyzed for their mechanisms of action through cell morphology assessments and molecular docking studies. mRNA expression levels were also evaluated to confirm the findings. RESULTS: The results showed that 3β-glucose sitosterol exhibited the most promising cytotoxic activity with IC CONCLUSION: Based on the results, β-sitosterol and its derivatives, particularly 3β-glucose sitosterol, show as the most promising potential adjuvant therapy for hormone-positive breast cancer.