The GAL4/UAS system is extensively used for targeted gene expression in Drosophila, but the strength of the GAL4 drivers and their effects on target genes are rarely quantified. Quantitative information about the strength of the perturbations introduced by the GAL4/UAS system would further expand the usefulness of the GAL4/UAS system in studying gene functions and developmental processes. We have developed an assay to determine the relative level of expression for target genes tagged with green fluorescent protein (GFP). Our assay enables the relative quantitation of fluorescent proteins within specific cell types and developmental time windows in living eggs/embryos, and permits the analysis of samples from a broad expression range. We illustrate the assay using a panel of four GAL4 drivers and three UAS responder lines in Drosophila oogenesis, discuss the issues associated with the interpretation of the quantitative data, and correlate our results with the analysis of the GAL4/UAS system at the transcript level. The imaging-based strategy described here can be used to quantify other GAL4 drivers in Drosophila and other organisms.