The present study reports the development of L-proline conjugated chitosan scaffold for wound healing appli-cation. Proline plays a beneficial role in collagen synthesis, and as a biochemical, it has the potential to modulate wound healing. In this regard, amino acid L-proline was conjugated onto chitosan, and the scaffolds were syn-thesised. FTIR and NMR analysis confirmed amino acid conjugation. The prepared scaffold was characterized by studies such as swelling, dissolution, tensile strength, porosity, water-vapor transmission rate and in-vitro healing properties. Cell viability assay showed that the scaffold has no cytotoxicity against the L929 and HaCaT cells. The in-vitro wound healing potential of the scaffold by scratch wound assay on the L929 cell line showed 53.35 +/- 2.3 %, 72.96 +/- 2.2 %, and 50.89 +/- 0.3 % wound closure for CS-P 200, CS-P 400 and CS-P 600, respectively when compared to native CS scaffold (38.86 +/- 1.6 %). A similar observation was found with HaCaT cells too. The studies showed that the modified scaffold promotes collagen deposition from fibroblast cells. These findings suggest that scaffold cues remodel the wound microenvironment for a better wound-healing state, and the L-proline conjugated scaffold may have considerable potential as a wound dressing to improve wound healing.