We use a microfluidic method to estimate the water permeability coefficient (p) of membranes. As model lipid membranes we employ giant unilamellar vesicles (GUVs) composed of palmitoyloleoyl phosphatidylcholine and cholesterol (10 mol%). We have developed a microfluidic device with multiple chambers to trap GUVs and allow controlled osmotic exchange. Each chamber has a ring-shaped pressure-controlled valve which upon closure allows isolation of the GUVs in a defined volume. Opening the valves leads to a rapid fluid exchange between the trapping region and the microchannel network outside, thus allowing precise control over solution concentration around the GUVs contrary to other experimental approaches for permeability measurements reported in the literature. The area and volume changes of individual vesicles are monitored with confocal microscopy. The solute concentration in the immediate vicinity of the GUVs, and thus the concentration gradient across the membrane, is independently assessed. The data are well fitted by a simple model for water permeability which assumes that the rate of change in volume of a GUV per unit area is linearly proportional to concentration difference with permeability as the proportionality constant. Experiments of GUV osmotic deflation with hypertonic solutions yield the permeability of POPC/cholesterol 9/1 membranes to be p = 15.7 ± 5.5 μm s-1. For comparison, we also show results using two other approaches, which either do not take into account local concentration changes and/or do not resolve the precise vesicle shape. We point out the errors associated with these limitations. Finally, we also demonstrate the applicability of the microfluidic device for studying the dynamics of vesicles under flow.