Benefits extended by probiotics have increased the consumption of probiotics which leads scientist to isolate and characterize new probiotic strains. This necessitates/imposes the need for safety assessment of the newly discovered probiotic cells on normal human cells. This protocol explains the measurement of cytotoxicity of probiotics considering the DNA replication as a tool. Actively growing cells are incorporating nucleotides in the S phase of cell cycle during the process of replication. If the cells are not viable, they won’t be able to replicate. 5-ethynyl-2′-deoxyuridine (EdU) is thymine nucleoside analog which can be incorporated in the newly synthesized strand of the DNA and the ability of its terminal alkyl group to react with azide dye under the presence of copper as a catalyst generate fluorescence which can be determined using fluorescence microscope or flow cytometry.