Wound healing is a fundamental physiological response to tissue injury that results in restoration of tissue integrity with optimal functions. It is characterized by the formation of granulation tissue and synthesis of major connective tissue components through the coordinated interplay of many extracellular matrix (ECM) proteins, growth factors and cells. Several wound healing biomaterials are available for various types of wounds with varied outcomes and efficacy. Amnion based scaffolds find a range of applications in regenerative medicine and there is a growing demand for quality amnion material for various therapeutic purposes. In this study, a new novel enzymatic process is developed for separation as well as processing of the membrane to improve its properties to cure both acute and chronic wounds. As the amnion is processed enzymatically using non collagenolytic protease, the noncollagenous antigenic components are removed whereas collagen proteins are preserved in the placental membrane. The processed amnion is envisaged to have high compatibility upon application for healing wounds as it contains collagen protein contents which are non-antigenic in nature. The enzymatically separated amnion membrane is characterized for its biophysical and cell viability or toxicity properties along with the amnion separated by conventional method and the characteristic studies suggest that the processed amnion materials are having desirable features for acting as a wound healing scaffolds.