In this study, the microscale thermophoresis (MST) method was applied to investigate the interaction of deferiprone with biometals (Fe3+, Cu2+, Zn2+, Co2+, Ni2+, Mn2+, Mg2+, and Ca2+). The MST results indicated significant interactions of deferiprone with Fe3+, Cu2+, Zn2+, Co2+, and Ni2+. The data fitted to the Hill model shows stoichiometry more than 1:1. No binding was observed for deferiprone with Mn2+, Mg2+, and Ca2+. Electrospray ionization mass spectrometry confirms the results obtained by MST indicating stable molecular ion peaks for the complexes of deferiprone with either Fe3+, Zn2+, Cu2+, Co2+, Ni2+, and Mn2+, but no binding was observed for deferiprone with either Ca2+ and Mg2+ under a gas phase state. MST shows a fast and simple approach to study the binding of deferiprone to different essential metal ions. Moreover, the complexes were stable for investigation by mass spectrometry under a soft ionization technique like used electrospray ionization, which aided to confirm binding stoichiometry.