The advent of fluorescent proteins has revolutionized signaling research, shifting focus from biochemical assays to analysis of live cells, organized tissues and even entire organisms. Modern applications of fluorescent proteins go beyond their use as specific markers of cells or tissues, allowing the researcher to visualize intracellular translocations as well as biochemical reactions. In this mini-review, we summarize the properties of a variety of fluorescent proteins, their detection using fluorescence microscopy and flow analysis, as well as their basic and more advanced applications, including fluorescence resonance energy transfer (FRET) to study signaling dynamics.