This study aimed to isolate Neferine from Nelumbo nucifera seeds and evaluate its cytotoxic effects on cancer cell lines, particularly SK-N-MC and HepG2 cells. Neferine was successfully isolated using a combination of hot methanol extraction, solvent partitioning, and flash column chromatography. The purity of the isolated compound was confirmed through chromatographic analysis. Cytotoxicity assessment using the MTT assay revealed dose-dependent decreases in cell viability for both Neferine and the reference standard cisplatin against SK-N-MC cells, with Neferine demonstrating significant cytotoxic effects even at lower concentrations. Cell cycle analysis and flow cytometry showed alterations in cell cycle distribution and fluorescence characteristics upon treatment with Neferine, comparable to those induced by Cisplatin, suggesting potential cell cycle arrest or delay at the G2/M checkpoint. Apoptosis investigation using Annexin V FITC/PI analysis further supported the cytotoxic effects of Neferine, with increased levels of apoptosis or necrosis observed in Neferine-treated HepG2 cells. These findings highlight the promising therapeutic potential of Neferine as an anti-cancer agent, warranting further mechanistic studies and clinical investigations.