Protein interactions with ligands or other proteins are controlled by a complex array of intermolecular forces. Although the interaction energies and intermolecular forces which contribute to the stabilization of the protein complex can be inferred indirectly from thermodynamic and kinetic approaches or be calculated with molecular simulation, recent progress in atomic force microscopy (AFM) has made it possible to quantify directly the ranges and magnitudes of the interaction forces between protein and other molecules. AFM has proved its value not only for resolving the topographical structure of protein samples, but also for probing the forces that control protein interactions or mechanical properties of proteins under physiological conditions. The objective of this review is to describe the uses of AFM in the determination of the forces that control biological interactions, focusing especially on protein-ligand and protein-protein interaction modes. We first consider measurements of the specific and the nonspecific forces that jointly control protein interactions. The review then indicates the theoretical background of AFM force curves and presents the great variety of force measurement modes that can be performed with this technique. In addition, some of the most recent studies in determining the unbinding forces and mechanical properties of proteins with AFM are reviewed and the available theoretical aspects necessary for the comprehension of the experiments have been provided.