The ability to create cell-derived decellularized matrices in a dish gives researchers the opportunity to possess a bioactive, biocompatible material made up of fibrillar proteins and other factors that recapitulates key features of the native structure and composition of in vivo microenvironments. By using cells in a culture system to provide a natural ECM, decellularization allows for a high degree of customization through the introduction of selected proteins and soluble factors. The culture system, culture medium, cell types, and physical environments can be varied to provide specialized ECMs for wide-ranging applications to study cell–ECM signaling, cell migration, cell differentiation, and tissue engineering purposes. This chapter describes a procedure for performing a detergent and high pH-based extraction that leaves the native, cell-assembled ECM intact while removing cellular materials. We address common evaluation methods for assessing the ECM and its composition as well as potential uses for a decellularized ECM.