Native mass spectrometry (native MS), performed under non-denaturing conditions, is one of pivotal analytical techniques for understanding the functions of biological macromolecules, including proteins and nucleic acids. By enabling the ionization of intact complexes formed through non-covalent interactions —hydrogen bonds, electrostatic interactions, and hydrophobic interactions— in aqueous solutions, native MS allows for their mass determination. This provides crucial insights into biologically relevant specific interactions. The present article highlights the author’s studies focused on determining the stoichiometry of macromolecular complexes and developing methodologies for characterizing specific protein–ligand interactions. These studies demonstrate the power of native MS in revealing molecular recognition mechanisms and providing information on the higher-order structure of macromolecules, both of which are essential for understanding diverse biological processes.