Some proteins undergo conformational changes when they bind their cognate ligand. This conformational change can be coupled to changes in the local environment of the chromophore of green fluorescent protein (GFP) to create genetically encoded sensors for those ligands. The periplasmic binding proteins of bacteria bind diverse classes of molecules and have been made into sensors for a number of neurotransmitters and metabolites. Here we describe protocols for using these sensors to quantify analytes in complex solutions in vitro and how to use them in cell culture-based assays to observe fluctuations in their concentration over time, from the millisecond existence of neurotransmitters to the consumption of cytosolic metabolites over the course of hours.