Probiotics are valued for their health-promoting properties, but a comprehensive safety assessment is essential before their integration into food and pharmaceutical applications. Cytotoxicity evaluation is a fundamental step in probiotic research, ensuring that bacterial strains do not exert adverse effects on mammalian cells. Cell viability assays, including the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, trypan blue exclusion assay, lactate dehydrogenase release assay and, carboxyfluorescein diacetate staining assay (CFDA) serve as reliable in vitro methods for assessing probiotic cytotoxicity. These assays provide critical insights into cell membrane integrity, metabolic activity, and overall cell survival. Human intestinal epithelial cell lines (e.g., Caco-2, HT-29) and immune-responsive macrophage models (e.g., RAW 264.7) are commonly used in cell viability assays. In addition, fluorescent method like CFDA has also been explored to determine the cytotoxicity of the probiotic bacteria in various cell lines. Additionally, fluorescent-based methods, such as those utilizing 5(6)-Carboxyfluorescein diacetate (CFDA), have been employed to assess the cytotoxicity of probiotic bacterial strains across different cell lines, enabling sensitive detection of compromised cell membrane function and viability. This chapter provides a comprehensive overview of methodologies for assessing probiotic cytotoxicity, emphasizing their importance in ensuring probiotic safety and efficacy before pre-clinical and clinical applications.