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Protein translocation is a universal event shared by most cell signalling pathways to transmit signals between cell compartments. In recent years, the use of new fluorescence microscopy technologies combined with fluorescent probes—most often fluorescent proteins—and image analysis software has allowed the visualization and extensive analysis of such dynamic events in the context of the living cell. This review article focuses on emerging fluorescence approaches that tackle live cell protein translocation in the image-free context. Such methods are based on either protein–protein interactions or analysis of spatial diffusion of proteins by fluorescence intensity measurements. The potential benefits of intensity measurement on global cell populations versus image analysis of heterogeneous cell sample are discussed in the context of drug discovery applications. [Copyright &y& Elsevier]