Determining the transepithelial electrical resistance (TEER) is a widely used method to functionally analyze tight junction dynamics in cell culture models of physiological barriers. Changes in temperature are known to have strong effects on TEER and can pose problems during the process of TEER measurements in cell culture vessels, complicating comparisons of TEER data across different experiments and studies. Here, we set out to devise a strategy to obtain temperature-independent TEER values based on the physical correlation between parameters such as TEER, temperature, medium viscosity and pore size of the cell culture inserts. By measuring the impact of temperature and different electrode types on TEER measurements on Caco-2 and HPDE (normal human pancreatic ductal epithelium) monolayers, we were able to derive a mathematical method that is suitable for the correction of TEER values for temperature changes. Applying this method to raw TEER values yields temperature-corrected TEER (tcTEER) values. Validity of tcTEER was demonstrated by showing a direct correlation with permeability of monolayers as determined by flux of RITC dextran. Taken together, the mathematical solution presented here allows for a simple and accurate determination of paracellular permeability independent of temperature variation during the process of TEER recording.