Probiotics are widely recognized for their health benefits; however, the potential production of cytolysins by certain strains raises safety concerns. Cytolysins, as pore-forming toxins, compromise cellular membranes, leading to host cell lysis and tissue damage. Strains such as Enterococcus faecalis pose potential risks, particularly when used in therapeutic or commercial applications. This chapter highlights in vitro cytotoxicity assay as a critical tool for assessing cytolysin production and evaluating the pathogenic potential of microorganisms. Comprehensive in vitro assessments are essential to mitigate the risks associated with cytolysin-producing strains. Cytotoxicity assays involve exposing cultured mammalian cells to bacterial supernatants, purified cytolysins, or other test samples to quantify toxic effects such as cell death, membrane disruption, and metabolic alterations. Methods like MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Lactate dehydrogenase (LDH) release, and fluorescence-based assays provide quantitative insights into cell viability, while advanced methods such as flow cytometry offer detailed characterization of cytolysin-induced cellular damage. These tools form the foundation for evaluating the safety and pathogenicity of microorganisms, particularly those with potential applications in probiotics and therapeutics.