In cancer research, it is an urgent need in the obtainment of a simple and reproducible model that mimics in all the complexity the pathological microenvironment. Specifically, the will to improve the overall survival of young patients affected by rhabdomyosarcoma compels researchers to develop new models resembling the multifaceted environment of the pathology to deeply study the disease under novel and different aspects. To this end, we developed a decellularization protocol for alveolar rhabdomyosarcoma (ARMS) able to maintain the three-dimensional structure. The attained extracellular matrix (ECM) can be used as 3D in vitro model suitable to both recapitulate the in vivo cancer microenvironment, and also for drug testing. Here, we first describe a detergent-enzymatic method and then we analyze the decellularization efficiency and the scaffold proteins.