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Intracellular signaling and cell-to-cell communication depend on the coordination of numerous signaling events, and this large flow of information has to be properly organized in space and time. Common and critical to all of these processes and the ultimate cellular response is the correct spatial distribution of signaling components and their targets. This fundamental concept applies to a large number of signaling processes. It is frequently important to quantify the localization of signaling molecules within different cellular compartments to detect subtle changes or to define threshold levels of signaling molecules in a certain location that are necessary to trigger subsequent events. Of particular importance is the separation of nuclear and cytoplasmic events, and sensitive methods are required to measure their contribution to signal transduction. Procedures described here allow the quantification of fluorescence signals located in the nucleus, cytoplasm, or at the nuclear envelope. The methods rely on high-throughput imaging equipment, confocal microscopy, and software modules that measure the fluorescence intensity in the compartment of interest. We discuss the rationale for selecting the appropriate equipment for image acquisition and the proper software modules to quantify fluorescence in distinct cellular compartments. Initially, high-throughput technology for high-speed image acquisition was developed for multiwell plates. We adapted high-throughput technology for image acquisition for cells grown on cover slips. Images of higher spatial resolution along the z axis were acquired by confocal microscopy. For subsequent analyses, the choice of appropriate software modules is critical for rapid and reliable quantification of fluorescence intensities.