Urease is an extracellular enzyme in various bacteria fungi, and plants that plays a crucial role in the pathogenicity of various microorganisms. Helicobacter pylori, for example, uses urease to hydrolyze urea. This produces ammonia, which increases the pH of its environment and allows the bacterium to survive in the acidic gastric mucosa, contributing to the development of gastric ulcers (Graham, Miftahussurur, J Adv Res 13:51–57, 2018). Many human pathogenic fungi, including Cryptococcus neoformans, Candida albicans, Coccidioides immitis, Histoplasma capsulatum, Sporothrixschenckii and species of Trichosporon also exhibit urease activity (Feder et al, FEBS J 282:1406–1418, 2015; Cox et al, Infect Immun 68:443–448, 2000). In C. neoformans, urease production during infection leads to the formation of ammonia, which alkalizes the tissue environment and contributes to tissue damage. This enzymatic activity has been linked to the ability of C. neoformans to cross the blood-brain barrier in mouse models of cryptococcosis and promotes invasion of the central nervous system (Toplis et al, FEMS Yeast Res 20:foaa031, 2020). Urease is a metalloenzyme that catalyzes the hydrolysis of urea to ammonia and carbon dioxide, increasing the pH of the surrounding medium. The pH shift can be visually detected by a color change of the phenol red indicator from light orange (pH 6.8) to pink (pH 8.1). Fungi with high urease activity usually cause the medium to turn pink within 24 h, and the intensity of the pink coloration correlates directly with the degree of urease activity (Cox et al, Infect Immun 68:443–448, 2000). This chapter describes a detailed protocol for measuring urease activity in pathogenic fungi, using urea agar plates as the medium for this assay. In this method, the appearance of a pink zone around the fungal colonies on the urea agar serves as an indicator of urease activity. The size and intensity of the pink zone can be quantitatively assessed to measure the level of urease production. This chapter describes a protocol for measuring urease activity in fungi.