Our research focuses on the elucidation of the PRPF31 gene mutation's impact on retinal cells, specifically within the context of Retinitis Pigmentosa (RP), employing human-induced pluripotent stem cells (hiPSCs). These hiPSCs, sourced from a patient with severe RP harboring the PRPF31 mutation and a healthy control, were differentiated into retinal organoids (ROs) using a refined protocol. The study's methodology encompassed a range of techniques including flow cytometry for assessing pluripotency, immunofluorescence and qPCR for progenitor and photoreceptor-specific markers, microelectrode array (MEA) recordings to gauge electrophysiological activity, and single-cell RNA sequencing (scRNA-Seq) to dissect the complexity of retinal cell populations and their gene expression patterns.The resulting ROs from the PRPF31 mutation displayed a phenotype that closely mirrors the progression of human RP, characterized by the gradual degeneration of rod photoreceptors, subsequently impacting cone cells. MEA analyses provided critical insights into the mutation's functional implications, revealing marked changes in electrophysiological activity. Furthermore, the scRNA-Seq examination shed light on previously undetected gene expression patterns, enhancing our understanding of the mutation's nuanced molecular effects and the inherent diversity within the retinal cell populations.In conclusion, this study, utilizing hiPSC-derived ROs, successfully simulates the progression of RP as influenced by the PRPF31 mutation. The combination of disease modeling with advanced analytical techniques like MEA and scRNA-Seq underlines the potential of these in vitro models. They represent a significant advancement in developing and evaluating targeted therapies and organic retinal prostheses, offering a promising avenue for personalized medicine in treating complex inherited retinal disorders.