Cytolysin proteins, produced by certain bacterial strains, are pore-forming toxins that compromise host cellular membranes, causing cell lysis and tissue damage. While probiotics are widely celebrated for their health benefits, the safety of some strains, such as Enterococcus faecalis, raises concerns due to the potential production of cytolysins. These toxins pose significant risks to vulnerable populations, necessitating rigorous safety evaluations of probiotic strains. In vitro assessment of cytolysin proteins using enzyme-linked immunosorbent assay (ELISA) offers a sensitive and specific method for quantifying these toxins. This chapter provides a detailed overview of the ELISA-based detection of cytolysin proteins, emphasizing its role in evaluating the pathogenic potential of bacterial strains to ensure their safety in therapeutic and commercial applications. The comprehensive evaluation of cytolysin production through ELISA contributes to safeguarding public health by mitigating risks associated with cytolysin-producing strains. This chapter highlights the need for comprehensive evaluations of probiotic strains to mitigate risks and safeguard public health, providing a detailed overview of detection of cytolysin proteins by ELISA.