Cytolysin proteins, produced by certain pathogenic bacterial strains such as Enterococcus faecalis, are pore-forming toxins that play a significant role in bacterial virulence. These toxins disrupt host cell membranes, leading to cell lysis and tissue damage, which can contribute to the pathogenicity of the bacteria. The potential health risks associated with cytolysin-producing bacteria, particularly in therapeutic and probiotic applications, underscore the need for rigorous safety evaluations. Western blotting is a powerful and reliable molecular technique for detecting and characterizing cytolysin proteins. This chapter provides a detailed protocol for the application of Western blotting to detect and assess cytolysin production in bacterial strains. Thus, by using Western blotting, researchers can assess cytolysin production in bacterial strains, contributing to the safe use of probiotics and ensuring public health safety.