Retinal pigment epithelium (RPE) stress and injury often leads to epithelial to mesenchymal transition (EMT), in which RPE cells lose its cobblestone morphology and acquire more motile and spindle-shaped fibroblast phenotype. RPE dysfunction due to acquired EMT phenotype have been implicated in a number of retinal diseases such as proliferative vitreoretinopathy (PVR), diabetic retinopathy (DR), neovascular (“wet”) and atrophic (“dry”) age-related macular degeneration (AMD). We investigated distinct temporal protein expression changes and signaling events that occur during enzymatically dissociated hRPE EMT model, as well as those that occur up to forty-eight hours after EMT onset. Further, we compared analysis on altered proteome profiling with malignancy associated EMT and AMD models. Together, proteome profiles provide a comprehensive RPE EMT resources for understanding molecular signaling events, associated biological pathways, that underlie RPE-EMT onset and further identifying chemical modulators that could potentially inhibit RPE EMT.