Study objective: Cell volume and water permeability measurements in cultured mammalian cells are typically conducted under a light microscope. Many of the employed approaches are not applicable to study confluent epithelial cell monolayers. We present here an adaptation of a calcein quenching based approach for a plate reader. Methodology: A standard curve of fluorescence intensities at equilibrium has been recorded, following shift from 285mOsmol to a series of altered extracellular osmolyte concentrations, ranging from final concentrations of 185mOsmol to 585mOsmol, by changing buffer D-mannitol concentrations. Likewise, according average cell volumes have been measured in suspension in a coulter counter (particle sizing device). Summary: We find that, while calcein fluorescence intensity changes linearly with extracellular osmolyte concentration, cell volume changes follow an exponential function. Based on these measurements we have derived an equation that facilitates to model cell volume changes based on fluorescence intensity changes. We have utilized the method to study the role of carboxyl-terminal Aquaporin (AQP)-2 phosphorylation sites, which are known to affect AQP2 membrane trafficking, in heterologous type I MDCK cells. We find enhanced water permeability in cells expressing AQP2 S256D or AQP2 S269D mutant AQP2 protein, when compared to cells expressing the AQP2 wildtype isoform. Conclusions: We propose that the method can be applied to study AQP function and more generally to study cell volume changes in adherent cell lines. Furthermore the method is suitable for identification of AQP inhibitors in chemical compound libraries.