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Tightly controlled spatiotemporal specificity of gene expression is intrinsic to developmental and adaptation responses of living systems throughout the kingdoms. Forward genetic screens employing well-characterized reporter lines can be used to identify as yet unknown genetic factors driving specific aspects of individual regulatory pathways. However, such screens are demanding with respect to data acquisition and analysis from thousands of mutant lines. Here, we describe a method that allows screening of a mutagenized GUS reporter line in Arabidopsis using an automated microscopy imaging system as a tool for rapid and efficient identification of mutants with modified expression profile for a gene of interest.