Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence probe at the end of trans-membrane helix 6 and reconstituted in phospholipid nanodiscs tethered to a microscope slide. Individual receptor molecules are then monitored over time by single-molecule total internal reflection fluorescence microscopy, revealing spontaneous transitions between inactive and active-like conformations. The assay provides information on the equilibrium distribution of inactive and active receptor conformations and the rate constants for conformational exchange. The experiments can be performed in the absence of ligands, revealing the spontaneous conformational transitions responsible for basal signaling activity, or in the presence of agonist or inverse agonist ligands, revealing how the ligands alter the dynamics of the receptor to either stimulate or repress signaling activity. The resulting mechanistic information is useful for the design of improved GPCR-targeting drugs. The single-molecule assay is described in the context of the β2 adrenergic receptor, but can be extended to a variety of GPCRs.