Proteases are ubiquitous in biosystems where they have diverse roles in the biochemical, physiological, and regulatory aspects of cells and organisms. Proteases represent the largest segment of the industrial enzyme market where they are used in detergents, in food processing, in leather and fabric upgrading, as catalysts in organic synthesis, and as therapeutics. Microbial protease overproducing strains have been developed by conventional screening, mutation/selection strategies and genetic engineering, and wholly new enzymes, with altered specificity or stability, have been designed through techniques such as site-directed mutagenesis and directed evolution. Complete sequencing of the genomes of key Bacillus and Aspergillus workhorse extracellular enzyme producers and other species of interest has contributed to enhanced production yields of indigenous proteases as well as to production of heterologous proteases. With annual protease sales of about $1.5–1.8 billion, proteases account for 60% of the total enzyme market. Detergent proteases, with an annual market of about $1 billion account for the largest protease application segment. Subtilisin Carlsberg and related subtilisin serine proteases represent the first generation of detergent proteases with pH optima of 9–10. The second generation, having higher pH optima (10–11) and greater temperature stability, is produced from alkalophilic strains including Bacillusclausii and B. halodurans. The third generation consists of detergent proteases whose active sites and/or stability have been modified by protein engineering. The principal applications of proteases in food processing are in brewing, cereal mashing, and beer haze clarification, in the coagulation step in cheese making, in altering the viscoelastic properties of dough in baking and in production of protein hydrolysates. In organic synthesis, proteases have application in synthesis and/or hydrolysis of peptide, ester, and amide bonds involving carboxylic acids and are effective tools for resolution of pairs of enantiomers. Proteases have applications in nutrition as digestive aids and in therapy in thrombosis and cancer treatment. Hyperproteolytic endogenous activity may play significant roles in abnormal physiological functioning as well as in microbial and viral pathophysiological conditions and this has created substantial momentum for development of protease inhibitors as therapeutic agents against disease-causing proteases.