Full osteochondral regeneration remains a major clinical challenge. Among other experimental cartilage regenerative approaches, decellularized cartilage (DCC) is considered a promising material for generating potentially implantable scaffolds useful as cartilage repair strategy. Bibliography is extensive, but decellularization methods are multiple and cartilage regenerative properties are limited. In this work, we focus on screening and comparing different decellularization methods, aiming to generate DCC potentially useful in biomedical context, and therefore, with biological activity and functional properties in terms of induction of differentiation and regeneration. Data indicate that enzymatic and detergents-based decellularization methods differentially affect ECM components, and that it has consequences in further biological behavior. SDS-treated DCC powder are not useful to be further processed in 2D or 3D structures, because these structures tend to rapidly solubilize, or disaggregate, in physiologic media conditions. Conversely, Trypsin-treated DCC powders processed to mechanically stable 2D films and 3D porous scaffolds, presumably due to partial digestion of collagens during decellularization. In vitro cell culture studies indicate that these structures are biocompatible and induce and potentiate chondrogenic differentiation. In vivo implantation of DCC derived 3D porous scaffolds in rabbit osteochondral defects induce subchondral bone regeneration and fibrocartilage tissue formation after implantation. Therefore, this work defines an optimal cartilage tissue decellularization protocol able to generate DCC powders processable to biocompative and bioactive 2D and 3D structures. These structures are useful for in vitro cartilage research and in vivo subchondral bone regeneration, while hyaline cartilage regeneration with DCC as implantable material remains elusive.