Cells have developed complex signal transduction cascades to respond to changes in their environment. Dynamic single-cell measurements are required to gain deeper insights into the regulatory mechanisms at work in these pathways and assess the variability in the cellular response. Microscopy is highly suited to deliver these datasets, because it is the only technique that enables us to follow the behavior of live cells while they activate a signal transduction cascade. In this chapter, we review a number of biosensors that have been developed to quantify diverse aspects of signal transduction, such as the sensing of metabolites, the activity of enzymes, or the synthesis of proteins.