NDI-Lyso is an anticancer agent targeting lysosomes, functioning through an enzyme-induced self-assembly (EISA) mechanism catalyzed by Cathepsin B. In cancer cell lysosomes, it forms rigid long fibers, promoting lysosomal swelling, lysosomal membrane permeabilization (LMP), and membrane disruption. This leads to non-classical caspase-independent apoptosis (Apoptosis). NDI-Lyso demonstrates significant selective anticancer activity across various cancer cell lines and drug-resistant cancer cells (IC50 approximately 10 μM), while exhibiting low toxicity to normal cells (IC50 > 60 μM).