To assess the wound healing activity of active fraction of stem extract of Argemone mexicana Linn. The methanolic extract of Argemone mexicana was obtained by cold maceration extraction and then different fractionation was obtained. Different antioxidant methods were performed. Isolation of components from ethyl acetate fraction and characterization was done by using different instrumental analysis. Herbal gel was prepared using the extract of various ingredients in different proportion. The evaluation of gel was done using the various sorts of parameters viz., appearance, pH, spreadibility. The evaluation of wound healing activity of active fraction of stem extract of plant was done on Wister albino mice using excision and incision wound models. Methanolic stem extract of Argemone mexicana Linn has shown profound presence of phytochemical constituents than other extracts. From the TLC it was found that the compounds isolated from ethyl acetate fraction show fluorescence at longer wavelength (360 nm). And from Rf value that is 0.33 and 0.66 value it was predicted that compound 1 (RK2A) and compound 2 (RK2B) may be berberine and sanguinarine or its analogue. Antioxidant activity of ethyl acetate fraction was found to be good. From the analgesic activity, anti-inflammatory activity it was found that ethyl acetate fraction having those properties which can be supportive study for wound healing. The ethyl acetate fraction of Argemone mexicana Linn treated wounds were found to be faster and rate of wound contraction was significantly increases as compared to control wounds(p<0.001) while in incision wound model tensile strength was good. Results indicate that the Argemone mexicana Linn stem is source of wound healing treatment.