Human pluripotent stem cells (hPSCs) is an alternative source of cells to develop production of therapeutic skin substitutes for cell therapy approach for chronic wound due to their proliferative and differentiation capacities. Since 2009, our group and others have developed a protocol allowing the differentiation of hPSCs into keratinocytes. For clinical use, in-process quality controls become essential to monitor the dynamic progression at each key step of the differentiation and serve as an indicator to manage the keratinocytes production. Keratin 5 (K5), encoded by KRT5 gene, is one of the key member of intermediate filament family expressed in basal keratinocytes. By creating a keratin 5 hPSC reporter cell line using CRISPR-Cas9 methodology, we propose to monitor the expression of keratin 5 on live cells during the differentiation process. For that, GFP gene associated to a Nucleus Localization Sequence, allowing the expression of the GFP in the nucleus, was directly introduce into KRT5 gene using CRISPR-Cas9 nuclease. After plasmid transfection and clone selection, hPCS were differentiated into keratinocytes. We observed the apparition of GFP expression from 15 to 20 days of differentiation corresponding to the expression of keratin 5 observed by immunostaining and flow cytometry analyses. These results confirmed a good correlation between GFP expression and K5 expression. This approach would allow continuous monitoring of cell production with a non-destructive in-process quality control to promptly adjust procedures and ensure the quality of the final product.