An experimental procedure associating both hydrogen/deuterium exchange mass spectrometry (H/D-MS) and mutagenesis was developed to identify the protein-binding region of small inhibitors targeting the motor domain of the human mitotic kinesin Eg5. All the tested inhibitors decrease the deuterium incorporation rate of the same peptides corresponding to the following secondary structure elements: loop L5/helix α2 (region Tyr125-Glu145) and strand β5/helix α3 (region Ile202-Leu227). Replacement of these two regions by the equivalent ones from N. crassa conventional kinesin heavy chain completely abolishes the modification of the deuterium incorporation rate by the inhibitors as well as their effects on the basal ATPase activity. The six tested inhibitors thus share a common binding site on Eg5. The strategy reported here allows the regions of a protein involved in ligand binding to be rapidly pinpointed and can be applied to other proteins and used as a general in vitro screening procedure to identify compounds targeting specific binding regions. Copyright © 2006 John Wiley & Sons, Ltd.