Electrospray ionization mass spectrometry (ESI-MS) binding studies between proteins and ligands under native conditions require that instrumental ESI source conditions are optimized if relative solution-phase equilibrium concentrations between the protein–ligand complex and free protein are to be retained. Instrumental ESI source conditions that simultaneously maximize the relative ionization efficiency of the protein–ligand complex over free protein and minimize the protein–ligand complex dissociation during the ESI process and the transfer from atmospheric pressure to vacuum are generally specific for each protein–ligand system and should be established when an accurate equilibrium dissociation constant (KD) is to be determined via titration. In this paper, a straightforward and systematic approach for ESI source optimization is presented. The method uses statistical design of experiments (DOE) in conjunction with response surface methodology (RSM) and is demonstrated for the complexes between Plasmodium vivax guanylate kinase (PvGK) and two ligands: 5′-guanosine monophosphate (GMP) and 5′-guanosine diphosphate (GDP). It was verified that even though the ligands are structurally similar, the most appropriate ESI conditions for KD determination by titration are different for each.