The migration of epidermal keratinocytes is the basis for skin re-epithelialization during wound healing. The in vitro scratch-wound assay using monolayers of primary human epidermal keratinocytes is a straightforward and effective method to assess their migratory capacity. The mechanical scratch of a confluent monolayer directly disrupts the adhesion of the keratinocytes to one another and to the underlying matrix, resembling the physical trauma of a wound in an in vitro assay. The keratinocytes will undergo an epithelial-to-mesenchymal transition, which will confer an ability to migrate toward each other to cover the gap by restructuring cell–cell and cell–extracellular matrix connections. However, a good scratch-wound method and protocol to ensure scratch reproducibility is essential, particularly when using primary cell cultures where donor variability may also impact results.