Collagen from different sources was isolated and designed as scaffolds to act as a three-dimensional substrate for culturing human skin fibroblasts, which can be used as dermal substitutes. The thermodynamic behavior of the scaffolds developed was analyzed through Differential Scanning Calorimetric (DSC) and Thermogravimetric analysis (TGA). Analysis by Fourier Transform Infrared Spectroscopy (FTIR) revealed the functional groups in the scaffolds and the mechanical stability of various scaffolds was assessed through tensile strength analysis. Human skin fibroblasts were cultured on the developed scaffolds to assess their cellular interaction and behavior, and the morphological characteristics of the cultured fibroblasts were evaluated using Scanning Electron Microscopy (SEM). The collagen scaffold exhibited unique features when developed from various sources and it was observed that cells could grow and proliferate well and spread as a monolayer in the reconstituted collagen scaffold.