We have combined saturation transfer difference NMR (STD NMR) with chemical shift imaging (CSI) and controlled concentration gradients of small molecule ligands to develop imaging STD NMR, a new tool for the assessment of protein-ligand interactions. Our methodology allows the determination of protein-ligand dissociation constants (KD) and assessment of the binding specificity in a single NMR tube, avoiding time-consuming titrations. We demonstrate the formation of suitable and reproducible concentration gradients of ligand along the vertical axis of the tube, against homogeneous protein concentration, and present a CSI pulse sequence for the acquisition of STD NMR experiments at different positions along the sample tube. Compared to the conventional methodology in which the [ligand]/[protein] ratio is increased manually, we can perform STD NMR experiments at a greater number of ratios and construct binding epitopes in a fraction (∼20%) of the experimental time. Second, imaging STD NMR also allows us to screen for non-specific binders, by monitoring any variation of the binding epitope map at increasing [ligand]/[protein] ratios. Hence, the proposed method does carry the potential to speed up and smooth out the drug discovery process.