Cell volume and water permeability measurements in cultured mammalian cells are typically conducted under a light microscope. Many of the employed approaches are time consuming and not applicable to study confluent epithelial cell monolayers. We present here an adaptation of a calcein quenching based approach for a plate reader. 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). 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 a carboxyl-terminus Aquaporin (AQP)-2 phosphorylation site, which is known to affect AQP2 membrane trafficking, in heterologous type I MDCK cells. We find that water permeability in cells expressing phosphorylation site mutants was in the following order: AQP2-S256D > AQP2 wildtype > AQP2-S256A. 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 it should be adaptable for AQP inhibitor screening in chemical compound libraries.