Perception of ligands in the extracellular space by transmembrane receptors initiates signal transduction. The conformation change of the receptor induces changes of intracellular signalling components, including altered cellular concentration, altered subcellular location, altered conformation and altered interacting partners. Biochemical approaches have yielded a lot of information about these processes. However, methods that are compatible with analysis of single living cells are often preferred, since cells are highly organized and their response is usually spatially heterogeneous. In addition, the study of signalling cascades requires high temporal resolution. Fluorescence imaging approaches meet these requirements. Moreover, imaging approaches can be combined with genetically encoded green fluorescent protein-based probes that have a high selectivity and sensitivity for the process/molecule of interest. Nowadays, many genetically encoded probes are available for visualizing signalling in living cells. This review is centred on a key regulator of cellular signalling, protein kinase C (PKC). We will discuss imaging approaches that are used for analysing the molecules involved in activation of PKC, visualizing the dynamics of the location of PKC, measuring the conformation of PKC and quantifying the activity of PKC. These approaches are of general interest since they can be applied to study the dynamics, conformation and activity of any protein in living cells.