In disease research, natural products have played a significant role in the process of drug discovery and development, and many mushroom species are considered small pharmaceutical factories producing hundreds of compounds with biological activity. Specifically, medicinal mushrooms harbor a repository of bioactive ingredients, encompassing polysaccharides, proteins, sterols, and alkaloids, which make them have anti-oxidant, anti-tumor, antiviral, and immunomodulatory properties. Hence, with the continuous development of modern medicine, the screening of natural products with specific biological activities from medicinal mushrooms has attracted the interest of many food and biopharmaceutical areas. Notably, cytotoxicity assays determine the ability of a substance to kill cells, which is important for identifying natural products that may have anti-cancer or other therapeutic properties. The MTT assay is one of the most popular tests to evaluate the activity of potential natural products. This chapter summarizes the experimental principle, material preparation, operational process, and matters for using the MTT assay to determine the cytotoxicity of natural products from medicinal mushrooms.