Skin injuries pose a significant medical challenge, particularly deep, extensive full thickness skin wounds that do not heal properly. These wounds often lack sensory nerves, which are crucial for skin sensitivity. The development of pre-innervated skin substitutes containing nerve cells offers a promising solution to enhance skin sensitivity in transplanted areas. For the production of such skin substitutes, direct transdifferentiation of fibroblasts into iNeurons using small molecules offers a fast and efficient approach. In this chapter, we provide a method of human dermis derived fibroblast transdifferentiation into iNeurons, and culture of iNeurons in a collagen type 1 hydrogel with a detailed protocol establishing a 3D model of pre-innervated skin substitutes. This hydrogel can further be used for development of pre-innervated dermal skin substitutes for clinical application, for investigation of disease mechanisms and drug screening.