The improvement of novel sequencing technologies is quickly transforming the scientific investigation and the therapeutic treatments of rare genetic diseases, which research is hampered by the limited cohort of diagnosed patients and by the difficulty to evaluate the effect of Variants of Unknown Significance (VUS). In silico predictors represent the gold standard of clinical entities for the interpretation of mutations identified through sequencing approaches. Unfortunately, a high number of them are classified as VUS, leading to wrong diagnoses and inadequate treatment of patients. To address this point, MITEseq (Mutagenesis by Integrated TilEs) is a novel saturation mutagenesis technique that allows to easily test thousands of potentially pathogenic protein variants in a single high throughput biological assay. As a proof of principle, we decided to apply it to mutagenize two regions (a portion of the DNA Binding Domain and the SAM domain) of P63, a transcription factor which, besides its oncogenic function, is mainly involved in skin development as regulator. To follow the biological activity of each generated variant, we set up a highly efficient conversion strategy from fibroblasts to keratinocytes-like-cells (through P63-KLF4 induction). By using the specific keratinocytes membrane antigen ITGβ4, we were able to separate the converted and non-converted cells, study the enrichment of each variant in the different populations and rank them according to their pathogenic effect. We finally aim to the development of a scRNA-seq based platform to expand such method to any other disease driving gene. Through this approach, we leverage a specific transcriptional signature to associate each cell (i.e. each variant) to a specific physiological or pathological activity.