Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is a biophysical tool for the characterization of protein dynamics following ligand binding (where the ligand is a small molecule, a peptide or another protein). HDX-MS has emerged as a rapid and sensitive approach to interrogate protein-protein interactions (PPIs) and transient protein folding states. In this presentation, we report the use of HDX-MS to characterize interactions of small molecules with PPI targets. Our results demonstrate that HDX-MS was able to identify the binding mode for PPI modulators, which was not accessible by conventional structure elucidation techniques. In addition to this, the characterization of the binding mode of phage display peptides by HDX-MS will be also discussed. These findings demonstrate that HDX-MS can validate and probe protein-ligand interactions with the concomitant impact on biology and drug discovery.