Cytolysins, pore-forming toxins that disrupt cellular membranes and cause host cell lysis, pose significant safety concerns, particularly in bacterial strains like Enterococcus faecalis. Evaluating the genetic potential for cytolysin production is crucial for determining the pathogenicity of these microorganisms. Polymerase chain reaction (PCR) is a precise and sensitive in vitro method for detecting cytolysin-associated genes, such as cylA and cylB. This technique enables targeted amplification of specific genetic markers, even in strains not actively expressing the toxin, making it an essential tool for screening bacterial strains for safety evaluations. The ability to detect cytolysin genes at the molecular level provides a more targeted and reliable approach compared to phenotypic assays, ensuring that strains with potential virulence are excluded from use. This chapter focuses on the application of PCR in identifying cytolysin genes, emphasizing its role in safeguarding therapeutic and commercial use of microbial strains by mitigating risks associated with cytolysin production.