Osteoarthritis is a major concern in the United States and worldwide. Current non-surgical and surgical approaches alleviate pain but show little evidence of cartilage restoration. Cell-based treatments may hold promise for the regeneration of hyaline cartilage-like tissue at the site of injury or wear. Cell–cell and cell–matrix interactions have been shown to drive cell differentiation pathways. Biomaterials for clinically relevant applications can be generated from decellularized porcine auricular cartilage. This material may represent a suitable scaffold on which to seed and grow chondrocytes to create new cartilage. In this study, we used decellularization techniques to create an extracellular matrix scaffold that supports chondrocyte cell attachment and growth in tissue culture conditions. Results presented here evaluate the decellularization process histologically and molecularly. We identified new and novel biomarker profiles that may aid future cartilage decellularization efforts. Additionally, the resulting scaffold was characterized using scanning electron microscopy, fluorescence microscopy, and proteomics. Cellular response to the decellularized scaffold was evaluated by quantitative real-time PCR for gene expression analysis.