Studying the toxigenicity of probiotic organisms is crucial for ensuring their safety and efficacy in promoting health. While probiotics are generally beneficial for gut health, they can also pose risks if they produce toxins or exhibit pathogenic traits. Probiotics should be safe for consumption, and assessing toxigenicity helps identify strains that could potentially produce harmful substances. Some probiotic strains might have pathogenic properties, and toxigenicity studies are essential to prevent such strains from being used in food or healthcare. This is especially important when working with novel or next-generation probiotic species, for which toxicity studies are often required prior to human clinical trials. These tests are also critical for protecting vulnerable populations, such as individuals with weakened immune systems or underlying health conditions. To ensure safety, in vitro safety tests can be employed to screen probiotic strains for traits like hemolytic activity or toxin production, excluding unsuitable candidates early in the development process. In addition, animal studies can help assess potential safety hazards, such as endocarditis, and determine the overall toxicity of a strain. This protocol is describing one of the in vivo methods (i.e., rabbit ileal loop method) widely used to determine the toxigenicity of probiotic organisms.