Cytolysins are potent bacterial toxins that disrupt host cell membranes, leading to tissue damage, inflammation, and cell lysis. These toxins are produced by various bacteria, including Enterococcus and Clostridium and play a critical role in bacterial pathogenicity. In probiotic strains, cytolysin production can contribute to their ability to outcompete harmful bacteria and also present risks, particularly in immunocompromised individuals. In vivo studies are essential to assess the safety and toxicity of cytolysins, offering a comprehensive understanding of their effects on the host organism. Mouse models, including immunocompetent and immunocompromised strains, are commonly used to evaluate cytolysin activity, immune modulation, and potential tissue damage. Key experimental approaches include toxicity assessments, cytokine profiling, and histopathological analysis. These studies help to determine the lethal dose (LD50) of cytolysins, monitor immune responses, and identify organ-specific damage. The findings from in vivo experiments are critical for evaluating the biosafety of probiotic strains and ensuring their safe use in therapeutic and dietary applications.