The MTT assay is a widely used colorimetric technique to assess cell viability, proliferation, and cytotoxicity. The assay is based on the ability of living cells to reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a yellow substrate, to purple formazan crystals via mitochondrial enzymes. This reduction is a reflection of cellular metabolic activity, which correlates with cell viability. In this protocol, cells are first cultured and treated with various compounds or conditions. Following treatment, MTT is added to the culture medium and incubated for several hours to allow the cells to convert MTT into formazan. The formazan crystals are then solubilized using DMSO, and the optical density (OD) of the resulting solution is measured using a microplate reader at 570 nm. The absorbance values are proportional to the number of viable cells, providing an indirect measure of cell proliferation and metabolic activity. This assay is particularly useful in drug screening, cytotoxicity studies, and in determining the effects of experimental treatments on cell health. The procedure described in this protocol provides step-by-step instructions for performing the MTT assay to ensure reliable and reproducible results, including cell preparation, treatment, MTT solution preparation, and data analysis.