Three-dimensional (3D) organotypic models are increasingly being used to study aspects of epidermal organisation and cutaneous wound-healing events. These are largely dependent on laborious histological analysis and immunohistochemical approaches. Here we outline a method for establishment of a versatile in vitro 3D organotypic skin equivalent that reflects murine epidermal organisation in vivo. The system is optically transparent and ideally suited to real-time analysis and integrated in situ imaging techniques. Moreover, the model permits the visualisation of epidermal regeneration following injury in real time, thereby facilitating avenues to explore distinctive modes of wound re-epithelialisation. The versatility of the model could help unravel molecular mechanisms underlying epidermal morphogenesis, assess novel therapeutic strategies and reduce animal experimentation in a non-invasive manner.