Zymography stands as a versatile technique pivotal in elucidating the effects of hydrolases on diverse biological substrates. The casein zymographic assay has emerged as a gold standard for assessing calpain activity, particularly in mammalian systems. This method allows for the simultaneous monitoring of calpain isoforms and calpastatin, the endogenous inhibitor, without the need for prior enzyme purification. Employing non-denaturing conditions on acrylamide gels embedded with casein, this protocol facilitates the digestion of casein by active calpains, resulting in distinct zones of proteolysis upon Coomassie blue staining. The differential gel mobility of calpain isoforms further aids in their differentiation. This methodology has been essential in investigating the impact of various factors, such as feeding strategies and postmortem treatments, on calpain activity in meat from different animal sources. This chapter focuses on the application of casein zymography and highlights the utility of this protocol in detecting calpastatin activity, overcoming previous limitations in its assessment.