The aim of this study is to develop a simple andcost-effective method for decellularization and preservation of human amniotic membrane (HAM) as a soft tissue replacement and a delivery system for stem cells. The HAM is decellularized (D) using new chemical and mechanical techniques. The decellularization scaffold is evaluated histologically and fully characterized. The cell adhesion and proliferation on the scaffold are also investigated and the biocompatibility of D tissues is evaluated in vivo. The histological studies reveal that the cells are successfully removed from the D tissue. The DNA extraction shows more than 95% cell removal (p= 0.001). The in vitro results indicate that the decellularisation process does not deteriorate the mechanical properties of the tissue, whereas it increases the in vitro biodegradation value (p< 0.05). In the D samples, there is no significant cytotoxicity, and no changes are found in the rate of cell proliferation (p> 0.05). Immunohistochemistry staining indicates that all the tested components remain unchanged within the D tissues. The count of inflammatory cells show that the decellularization process slightly increases the biocompatibility of the tissue after 7 days post-surgery. The results indicate that scaffold proves to be reproducible, rapid, and cost-effective, with a potential role for clinical application.