Polymer vesicles, so-called polymersomes, can be applied as carrier-systems and universal reaction compartments, due to the possibility to encapsulate guest molecules. Compared to common lipid vesicles, polymersomes show an increased stability and decreased membrane permeability. Control of the mass transport across the membrane is necessary for any application, requiring the precise knowledge of the permeability. So far, data on permeability coefficients of polymersomal membranes are scarce because commonly applied release assays are confronted with the challenge of high detection limits and alternative methods developed so far are either restricted to the use of a certain permeating molecule or rely on the use of nuclear magnetic resonance measurements. In contrast, an influx assay that is broadly applicable to hydrophilic molecules and does not involve specialized equipment was developed in this work, which is based on the passive diffusion of compounds into initially empty vesicles. The method is valid for hydrophilic molecules that show no membrane retention and, thus, do not accumulate within the membrane. Using this method, the permeability of polymersomes made of poly(2-methyloxazoline)(15)-poly(dimethylsiloxane)(68)-poly(2-methyloxazoline)(15) for seven model compounds was investigated under varying conditions. Permeability coefficients as low as 1.9 X 10(-14) cm s(-1) could be measured.