Metal ions, usually bound by various amino-acid side chains in proteins, play multiple roles in protein folding, conformational change, cellular communication, and catalysis. Ca(II) and Mg(II), abundant among biologically relevant cations, execute their cellular functions associated with the conformational change of bound proteins. They bind with proteins where carboxylic acid residues are dominant ligands. To develop mass spectrometry for mapping protein-binding sites, we implemented a new carboxyl group footprinter, benzhydrazide, and refined it with isotope encoding. The method uses carbodiimide chemistry to footprint carboxylic residues, whereby 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide activates a carboxyl group followed by nucleophilic attack by benzhydrazide forming a stable labeled product. We tested the effectiveness of isotope-encoded benzhydrazide by studying Ca2+ and Mg2+ binding of calmodulin, an EF-hand protein. The footprinting results indicate that the four active sites for metal-ion binding (EF hands I, II, III, and IV) and the linker region (peptide 78-86) undergo conformational changes upon Ca(II) and Mg(II) binding, respectively. The outcome is consistent with previously reported results and 3-D structures, thereby validating a new reagent that is more reactive and discriminating for specific amino-acid protein footprinting. This reagent should be important for locating metal-binding sites of other metalloproteins.