The application of cell-derived extracellular matrix (ECM) in tissue engineering has gained increasing interest because it can provide a naturally occurring, complex set of physiologically functional signals for cell growth. The ECM scaffolds produced from decellularized tissues contain high amounts of ECM substances, such as collagen, elastin, and glycosaminoglycans. These substances can provide cell adhesion sites and mechanically strong supports for tissue-engineered constructs. Thus, the implementation of an efficient decellularization method that can totally remove cellular materials while maintaining minimal disruption of ECM ultrastructure and content is critical. There are two main approaches where decellularized matrices are applied: i) using it as scaffolds per se or ii) combining it with other biomaterials to develop new and improved scaffolds. In this study, we aimed the second approach. For so, human adipose derived stem cells (hASCs) were cultured under basal conditions (αMEM supplemented with 10% FBS and 1% antibiotic/antimycotic) and under osteogenic conditions (basal medium supplemented with 100 nM dexamethasone, 10 mM b-glycerophosphate and 0.05 mM ascorbic acid). After 6 weeks of culturing, cells detached from the bottom of the well and created a kind of microtissue. These microtissues were decellularized, obtaining two different matrices, basal matrices and osteogenic matrices, with distinct features. In this sense, the second mentioned, osteogenic matrices, presented enriched features for bone tissue regeneration approaches (i.e. mineralization deposits). The efficiency of the decellularization process was validated using DNA quantification and H&E staining for nucleus observation. Additionally, scanning electron microscopy (SEM) was used to confirm the presence (osteogenic matrices) or absence (basal matrices) of mineralization deposits. Finally, the presence of collagen type I within the matrices was assessed by immunocytochemistry. In brief, we propose a class of raw materials that can be mixed with different biomaterials to address a variety of bone tissue engineering applications.