Chitosan is soluble in diluted acid solutions and can easily form films by casting. However, residual acid neutralization should be performed for biomedical applications what may compromise physical and mechanical properties of the films. Thus, plasticizers can be added to improve these properties. The aim of this study was to characterize morphological, barrier and mechanical properties, besides evaluate the in vitro cytotoxicity of sorbitol-plasticized and NaOH-Na2CO3 neutralized chitosan membranes for skin substitute application. Scanning electron microscopy, X-ray diffraction, water vapor permeability and mechanical tests were carried out to characterize the obtained membranes. Moreover, Vero cells were used for in vitro cytotoxicity evaluation. In this paper, we report a non-cytotoxic sorbitol-plasticized chitosan membrane with desirable properties for skin substitution, such as flexibility, water vapor permeability and high percentage of elongation Ver menos