Proteases, also known as proteinases or proteolytic enzymes, are a large group of hydrolases that catalyze the cleavage of peptide bonds in proteins to produce peptides and/or amino acids. Classification of proteolytic enzymes is based on three major criteria: type of reaction catalyzed, functional group of the active site, and type of molecular structure and evolutionary relationship among the various enzymes. According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, the proteolytic enzymes can be grouped into proteases and peptidases on the basis of their nature of attack. This chapter describes methods to estimate soil protease activity, utilizing two substrates: casein, essentially a nonspecific substrate, and N-benzoyl-L-argininamide (BAA), a typical substrate for trypsin-like enzyme. The assay is based on colorimetric estimation of products released by the protein and amide-hydrolyzing enzymes when soil is incubated with buffered solutions of casein and BAA, respectively.