The retinal pigment epithelium (RPE) is a specialized epithelium lying in the interface between the �neural retina and the choriocapillaris where it forms the outer blood–retinal barrier (BRB). The tight junctions (TJ)s expressed in the outer BRB control fluids and solutes that enter the retina and this sealing function, which is essential for the retinal homeostasis, is impaired in diabetic retinopathy. In this �chapter, we provide the methods to explore the function of the RPE barrier by measuring Transepithelial electrical resistance (TER) and paracellular permeability to dextran in cultures of ARPE-19 cells (an immortalized RPE cell line). A method for inducing a lesion mimicking which occurs in diabetic retinopathy is described. In addition, methods for assessing mRNA expression and protein content of the main TJ proteins (occludin, zonula occludens-1 [ZO-1]) are detailed. Finally, we provide the methods required for confocal immunofluorescence detection of the TJ proteins, as well as for assessing the capacity of ARPE-19 cells to retain their functional properties.