Probiotics are widely used nowadays for their prophylactic and therapeutic activities against different gastro-intestinal diseases and cancers. Probiotic organisms are consumed as live organisms. However, many reports indicated the cytotoxic effects of probiotic organisms against the normal human cells. Consequently, it is necessary to evaluate the cytotoxic effects of probiotics against the healthy human cells in order to propose safe therapeutic agent for consumption. With the purpose of assessing the cytotoxicity of probiotics on normal healthy human cells, current protocol explains the methodology of live and dead cells dual staining method using calcein-acetoxymethyl ester (Calcein AM) and propidium iodide (PI) stains to evaluate the cytotoxicity of probiotics against normal Human embryonic intestinal cell line (INT-407). Numerous commercial kits are also available for this live/dead dual staining which can be used for the cytotoxicity determination. This method utilizes the ability of live and dead cells to uptake the differential stains based on their membrane permeability and measures the uptake of dyes by measuring the fluorescence generated by different mechanisms utilized by live and dead cells such as metabolic reactions or interaction with the biomolecules inside the cells. The available protocol also explains cytotoxicity determination using fluorescence microscopy, fluorescence plate reader and fluorescence cytometry along with considerations for the selection of optical filters, optimization of dye concentration, etc.