AbstractMotivation: Quantifying variability in protein expression is a major goal of systems biology and cell-to-cell variability in subcellular localization pattern has not been systematically quantified.Results: We define a local measure to quantify cell-to-cell variability in high-throughput microscope images and show that it allows comparable measures of variability for proteins with diverse subcellular localizations. We systematically estimate cell-to-cell variability in the yeast GFP collection and identify examples of proteins that show cell-to-cell variability in their subcellular localization.Conclusions: Automated image analysis methods can be used to quantify cell-to-cell variability in microscope images.Contact: alan.moses@utoronto.caAvailability and Implementation: Software and data are available at http://www.moseslab.csb.utoronto.ca/louis-f/Supplementary information: Supplementary data are available at Bioinformatics online.