In vitro cell culture methods are used extensively to study cellular migration, proliferation, and differentiation, which play major roles in wound healing but the results often do not translate to the in vivo environment. One alternative would be to establish an ex vivo model utilizing human discarded skin to evaluate therapies in a more natural setting. The purpose of this study was to institute such a model by creating ‘wounds’ in the center of a piece of discarded skin and treating them with three different biomaterials: collagen, polyethylene glycol (PEG)-fibrin, or PEG-platelet free plasma (PFP). Explants were cultured for 14 days with supernatant and microscopy images collected every 3 days to assess cytotoxicity and epithelialization. After 14 days, the explants were fixed, sectioned, and stained for cytokeratin-10 (CK-10), alpha-smooth muscle actin (α-SMA), and wheat germ (WG). Compared to controls, similar levels of cytotoxicity were detected for 12 days which decreased slightly at day 14. The PEG-PFP hydrogel-treated wounds epithelialized faster than other treatments at days 6 to 14. A 6-8 cell layer thick CK-10+ stratified epidermis had developed over the PEG-PFP hydrogel and cells co-stained by WG and α-SMA were observed within the hydrogel. An ex vivo model was established that can be used practically to screen different therapies exploring wound healing.