The development and differentiation of stem cell-derived impermeable retinal pigment epithelium (RPE) with tight junctions (TJs) is a gradual process that is, at confluence, controlled by cell-to-cell contact. The objective of this study was to evaluate the use of electric impedance spectroscopy (EIS) to follow the maturation and development of barrier function in human embryonic stem cell-derived RPE (hESC-RPE). Barrier function was assessed using EIS, permeability measurements, and microscopic inspection in intact cells and following calcium sequestration with ethylene glycol tetraacetic acid (EGTA). The results showed that the cultures with the most mature morphology had the highest impedance and the lowest permeability values. The EIS of samples of high integrity fitted well to the equivalent model of a single RC circuit, whereas the semicircular shape of the Nyquist plots was distorted for samples of lower integrity. EGTA treatment resulted in lower impedance values and changes in the shapes of plots. Our results show that EIS-as a measure of overall maturity and integrity of the epithelium-is useful when evaluating the maturity of cell cultures. It is highly warranted in future transplantation therapies and in in vitro cell culture models in drug development.