Decellularization is the process of removal of native cells from tissue, leaving behind a three-dimensional (3D) ultrastructure of extracellular matrix (ECM) proteins while preserving the bioactivity and mechanics of the tissue. It offers a unique top-down approach for fabricating ECM based natural scaffold for tissue engineering application. Herein, this chapter presents the fabrication of decellularized scaffold employing different methods: whole organ perfusion, immersion and agitation, pressure gradient, and supercritical fluid. The decellularized scaffold aims to exploit the nature-designed 3D architecture, a successful platform technology, for creating scaffolding materials for tissue engineering and regenerative medicine.