Plant leaves are often used for both therapeutic and nutritional purposes as they are rich sources of bioactive secondary metabolites. Despite their benefits, they may be toxic to humans. This chapter provides an overview of in vitro cytotoxicity evaluation of leaf extracts using the MTT [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay to ascertain their safety and potential applications. The MTT assay is quick, easy, well-established, widely used, and allows many assays to be carried out simultaneously. It involves exposing NIH/3 T3 mouse embryonic fibroblast cell lines to water, methanol, ethanol, or hydroalcoholic leaf extract at various concentrations to measure the reduction of yellow MTT tetrazolium salts by active cells to insoluble purple formazan crystals. The crystals are then solubilized with dimethyl sulphoxide (DMSO) solution, and absorbance is measured by a microplate reading spectrophotometer at a wavelength of 570 nm and compared to the absorbance in a control solution. The concentration of the leaf extract required to achieve 50% growth inhibition (IC50) is used to assess the cytotoxicity of the extract.