All cells are different. Even isogenic cells can possess diverse shapes, reside in different cell-cycle stages or express various sets of proteins. These variations can modulate the cell response to environmental stimuli and thereby provide key insights into the regulation of signal transduction cascades. Fluorescence microscopy allows to visualize these differences and monitor in real-time the responses of live single cells. In order to observe key cellular events, fluorescent biosensors have been developed. Among many assays, relocation reporters play an important role since they enable the quantification of the signal transduction dynamics. Fluorescently tagged endogenous proteins, as well as synthetic constructs, have allowed the measurement of kinase activity, transcription factor activation, transcription and protein expression in live single cells.