G-quadruplexes (G4s) have become important drug targets to regulate gene expression and telomere maintenance. Many studies on G4 ligand binding focus on determining the ligand binding affinities and selectivities. Ligands, however, can also affect the G4 conformation. Here we explain how to use electrospray ionization mass spectrometry (ESI-MS) to monitor simultaneously ligand binding and cation binding stoichiometries. The changes in potassium binding stoichiometry upon ligand binding hint at ligand-induced conformational changes involving a modification of the number of G-quartets. We investigated the interaction of three quadruplex ligands (PhenDC3, 360A and Pyridostatin) with a variety of G4s. Electrospray mass spectrometry makes it easy to detect K+ displacement (interpreted as quartet disruption) upon ligand binding, and to determine how many ligand molecules must be bound for the quartet opening to occur. The reasons for ligand-induced conversion to antiparallel structures with fewer quartets are discussed. Conversely, K+ intake (hence quartet formation) was detected upon ligand binding to G-rich sequences that did not form quadruplexes in 1 mM K+ alone. This demonstrates the value of mass spectrometry for assessing not only ligand binding, but also ligand-induced rearrangements in the target sequence. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.