Abstract Background The clinical application of amniotic membrane wound healing products is limited partially due to handling challenges. Amnion-CollaGee sponge dressing (ACS) provides better alternatives to traditional therapy in facilitating wound healing. Objective To optimize the effectiveness of ACS as a healing agent in donor-site split-thickness skin graft (STSG) based on in vitro outcomes. Followed by comparing wound healing rates, pain sensitivity, and risks of infection between investigator ACS dressing with currently conventional Duoderm dressing (DD) via in vivo analysis. Materials and methods Seven male Sprague-Dawley rats underwent STSG harvesting, with 1x1cm grafts taken from both the right and left sides of the animals' backs. The wounds on the right side (Group 1) were dressed with ACS, while the wounds on the left side (Group 2) were dressed with DD. The donor site wounds were assessed on the seventh, fourteenth, and twenty-first days of the experiment. Primary outcomes measured were rate of healing, pain sensitivity, potential risks of infection. Secondary outcomes were lesion vascularity, pigmentation and histopathological features. Results The size of the wounds treated with ACS was statistically smaller compared to that treated with DD. On day 21st, both types of dressings showed complete healing, thus, contributing to better aesthetic value. Additionally, superior pain suppression was witnessed at ACS-treated graft sites compared to DD. Histopathological reports displayed better post-traumatic wound site inflammatory control, less tissue scarring, and abundant collagen growth in the ACS wing compared to the DD counterpart. Conclusion The biological ACS dressing appeared to facilitate rapid wound healing rates, reduce the risk of exposed wound infections, and enhance pain suppression in rodent models. ACS based dressing agents are future interventional breakthroughs in graft donor site wound management.