Fluorescent proteins (FPs) are quite versatile imaging probes and have been successfully employed in almost every biological discipline from microbiology to systems physiology. They are extremely useful as reporters for gene-expression studies in cultured cells, excised tissues, and whole animals. In this article, the discussion of FP applications will be focused on live cell microscopy techniques. In live cells, FPs are most commonly used to track the localization and dynamics of proteins, organelles, and other cellular compartments. FPs can also be used to assess protein-protein interactions in living cells through the use of fluorescence resonance energy transfer (FRET). This article provides some general tips for the practical aspects of using and imaging enhanced green fluorescent protein (EGFP) and newer members of the color palette, as well as quantitative imaging of FPs and imaging of several FPs at the same time. Finally, an overview is provided for the different types of biosensors that have been derived from FPs.