Probiotics are widely recognized for their health benefits, but the safety of certain strains remains a concern due to the potential production of cytolysins. These pore-forming toxins disrupt cellular membranes, leading to host cell lysis and tissue damage. Strains such as Enterococcus faecalis, while beneficial in some contexts, may pose risks to vulnerable populations when they produce cytolysins. Evaluating the pathogenic potential of these microorganisms is crucial for ensuring their safety in therapeutic and commercial applications. In vitro assessment methods for cytolysin production include hemolysis assays, molecular approaches like polymerase chain reaction, immunological techniques such as enzyme-linked immunosorbent assay, and proteomic analyses. Hemolysis assays, which involve observing red blood cell lysis on blood agar plates, serve as a primary screening tool to detect cytolysin activity. This chapter explores the methodology and applications of hemolysis assays in evaluating cytolysin production, emphasizing their role in the risk assessment of probiotic strains to ensure their safety for therapeutic and commercial applications. Comprehensive in vitro evaluations are essential for safeguarding public health while harnessing the benefits of probiotics.