In this paper a novel and easily applied method to measure the mass transfer resistance of the sterile closures (e.g. cotton plug) of shaken bioreactors is introduced. This method requires no investment in special equipment (e.g. an oxygen sensor) and can be performed with the materials usually available in typical laboratories. The method is based on the model of Henzler et al. (1986), which mechanistically describes mass transfer through the sterile closure of a shaken bioreactor based on diffusion coupled with Stefan convection. The concentration dependency of the multi-component diffusion coefficients is taken into account. The water loss from two equivalent shaken bioreactors equipped with sterile closures during several days of shaking is measured. One flask contains distilled water, the other a saturated salt solution. From the water evaporation rate in each of the two flasks, the new model presented calculates the relative humidity in the environment, the average diffusion coefficient of oxygen in the sterile closure ($D_{{\rm O}_2 }$), and the diffusion coefficient of carbon dioxide ($D_{{\rm CO}_2 }$). The diffusion coefficient of carbon dioxide ($D_{{\rm CO}_2 }$) only depends on the density and material properties of the sterile closure and not on the gas concentrations and is, therefore, an ideal parameter for the characterization of the mass transfer resistance. This new method is validated experimentally by comparing the diffusion coefficient of oxygen ($D_{{\rm O}_2 }$) to a measurement by the classic dynamic method; and by comparing the calculated relative humidity in the environment to a humidity sensor measurement. Biotechnol. Bioeng. 2007;98: 999-1007. © 2007 Wiley Periodicals, Inc.