Clotting of milk by coagulating enzymes is the first necessary step in the production of ripened cheese varieties. This chapter summarizes and interprets the latest findings on the manufacture and application of the most important types of coagulants. Starting with animal rennet from the abomasal mucosa of ruminants, genetically engineered chymosin, coagulants of microbial origin, and plant-derived clotting enzymes are discussed. Special emphasis is placed on the main analytical methods that are used to evaluate the performance of the coagulants, and the impact of these enzymes on proteolysis, cheese yield and quality.