Summary: We present an optimized protocol for guided differentiation of retinal pigment epithelium (RPE) cells from human-induced pluripotent stem cells (iPSC). De novo-generated RPE cells are mature, polarized, and mimic the cellular and molecular profile of primary RPE; they are also suitable for in vivo cell transplantation studies. The protocol includes an enrichment step, making it useful for large-scale GMP manufacturing. RPE cells produced following this protocol are appropriate for cell replacement therapy for macular degeneration and disease modeling.For complete details on the use and execution of this protocol, please refer to Surendran et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.