Sperm capacitation is the final sperm maturation step that occurs in vivo in the female oviduct, triggered in a response to ovulation. Physiologically, to achieve full fertilizing ability, sperm capacitation requires a series of events to occur. This includes ion fluxes, intracellular pH increase, signaling and metabolic pathway activation, membrane reorganization, cholesterol efflux, release of decapacitating factors from the sperm surface, and membrane protein redistribution, to name a few. Fertilizing ability is reflected in hyperactivated motility, enabling capacitated spermatozoa to detach from the oviductal epithelium and migrate to the oviductal infundibulum; zona pellucida binding affinity; and inducibility of acrosomal exocytosis. In vitro capacitation (IVC) can be achieved by incubating spermatozoa in capacitation-supporting media. Here we describe our laboratory protocols for IVC of porcine, bovine, and human spermatozoa.