Background and Aim: Individual herbs, according to Ayurveda, are insufficient to provide the intended medicinal effect. When it is adjusted as a multiple herb composition in a certain ratio, it provides a greater medicinal impact with less toxicity. This investigation is an attempt to evaluate wound healing activity of poly herbal formulation along with in-vitro and in-vivo pharmacological activity on Swiss albino rats. Experimental Procedure: Full skin defects were made in the dorsal region of rats in order to examine wound healing. The activity of the polyherbal formulation was carried out by in-vitro (hydroxyproline, collagen and hexosamine) and in-vivo (excision and burn) assays along with histopathological study. Silver sulfadiazine was used as standard drug for the experimental work of this project while ketamine hydrochloride (20mg/kg intramuscular) was used as anesthize agent. The use of 2 μg/ml and 4 μg/ml formulations resulted in substantial mobilization of keratinocytes and fibroblasts at the injury site, respectively. In case of in vitro activity, effect of polyherbal formulation on biochemical measures such as hydroxyproline, collagen and hexosamine turnover were enhanced compared with untreated group. Results: Polyherbal formulation containing herbal extract was applied on wound area on day 0, 3rd, 6th, and 9th and was found to be more significant. Burn wound area on last day was observed 26.30 ± 0.5 while surgically produced wound area was 13.666± 0.8. Conclusion: Finally, on the basis of above results, we can conclude that polyherbal formulation may be a breakthrough for the treatment of wound healing in future.