Protein–ligand interactions play an important role in many biological processes. Notably, membrane receptors are the starting point for a huge variety of cellular signal transduction pathways. Quantifying the binding affinity of a ligand for its transmembrane receptor is of great importance as it provides information on the potency of the ligand. We developed a new experimental procedure to determine binding affinities of ligands for their membrane receptors directly on intact single cells using super-resolution imaging. Dissociation constants were determined by titrating fluorophore-labelled ligand against cells expressing the target protein and applying single-molecule imaging.