Artificial dermal constructs, based upon collagen-glycosaminoglycan matrices (CGMs), provide new options in treating skin defects. However, their clinical effectiveness may be limited by cytotoxicity related to residual aldehydes left over from the manufacturing process. Although both chemical and dehydrothermal (DHT) cross-linking are used to produce CGMs, we hypothesize that optimized nonchemical cross-linking, using ultra-violet (UV) and DHT treatment combinations, may limit cytotoxicity without sacrificing mechanical strength. Porous CGMs were physically cross-linked using a combination of DHT and varying intensities of UV light. These were compared to glutaraldehyde cross-linked controls. Human keratinocytes were seeded in each matrix, and cellular proliferation measured using a microculture tetrazolium dye assay. A scoring system (based on the in vitro contraction rate, stiffness, and cellular growth of a small cylindrical specimen) was developed to assess the best overall physical cross-linking method. More cellular growth was observed in the 90-120 min UV cross-linked group than in the glutaraldehyde-treated group (p < 0.05). Stiffness was maximized after 0-30 min of UV cross-linking. On the basis of our scoring system, DHT combined with 45 min of UV cross-linking produced the best overall matrix in terms of cellular growth and physical durability. UV cross-linked collagen-based biomaterials could be a viable alternative for use in biological applications to eliminate glutaraldehyde-associated cytotoxicity.