Decellularized membranes (DM) were obtained from bovine amniotic membranes (BAM) using four different decellularization protocols, based on physical, chemical, and mechanical treatment. The new material was used as a biological scaffold for in vitro skin cell culture. The DM were characterized using hematoxylin-eosin assay, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR-ATR), and differential scanning calorimetry (DSC). The in vitro cytotoxicity of DM was evaluated using MTT. The efficacy of decellularization process was assessed through DNA quantification and electrophoresis. All the used protocols showed a high effectiveness in terms of elimination of native cells, confirmed by DNA extraction and quantification, electrophoresis, and SEM, although protocol IV removes the cellular contents and preserve the native extracellular matrix (ECM) architecture which it can be considered as the most effective in terms of decellularization. FTIR-ATR and DSC on the other hand, revealed the effects of decellularization on the biochemical composition of the matrices. There was no cytotoxicity and the biological matrices obtained were a source of collagen for recellularization. The matrices of protocols I, II, and III were degraded at day 21 of cell culture, forming a gel. The biocompatibility in vitro was demonstrated; hence these matrices may be deemed as potential scaffold for epithelial tissue regeneration.