Proteins are major drug targets, and drug-target interaction identification and analysis are important factors for drug discovery. Atomic force microscopy (AFM) is a powerful tool making it possible to image proteins with nanometric resolution and probe intermolecular forces under physiological conditions. We review recent studies conducted in the field of target protein drug discovery using AFM-based analysis technology, including drug-driven changes in nanomechanical properties of protein morphology and interactions. Underlying mechanisms (including thermodynamic and kinetic parameters) of the drug-target interaction and drug-modulating protein-protein interaction (PPI) on the surfaces of models or living cells are discussed. Furthermore, challenges and the outlook for the field are likewise discussed. Overall, this insight into the mechanical properties of protein-drug interactions provides an unprecedented information framework for rational drug discovery in the pharmaceutical field.