Conventional cancer therapies often harm healthy tissues due to their poor specificity, resulting in significant side effects that diminish patients’ quality of life. Parasporins, a group of non-hemolytic parasporal proteins produced by Bacillus thuringiensis, are known for their selective cytotoxicity toward cancer cells. Typically, these proteins require activation through physical or biochemical treatments that fragment them into multiple peptides of varying sizes, which are then tested as mixtures, without purification, against cancer cell lines. In this study, a purification strategy that isolates the protein without prior activation and evaluates the resulting cytotoxic mechanism is proposed. The purification consists of four steps: (1) crystal solubilization with Laemmli buffer, (2) size-based separation via SDS-PAGE, (3) electroelution of the target protein from the gel, and (4) dialysis to remove the elution buffer. From the B. thuringiensis AX isolate, four proteins ranging from ~20 to 60 kDa were recovered, but only AX-2 displayed cytotoxic activity toward MCF-7 breast cancer cells, while remaining non-hemolytic and non-toxic to normal cells (erythrocytes, PBMCs, and MRC-5 fibroblasts). Thus, AX-2 qualifies as a parasporin. AX-2 induces apoptosis in MCF-7 breast cancer cells without generating oxidative stress, and the observed cell death appears to initiate at the plasma membrane rather than through intracellular pathways.