This is the final published version. It was first made available by RSC at http://pubs.rsc.org/en/Content/ArticleLanding/2015/CC/c5cc02252e#!divAbstract. ; A rapid and simple equilibrium-binding assay mediated by ligand-induced fluorescence quenching of fluorophore-labelled G-quadruplex (G4) structures enabled quantitative interrogation of mutually exclusive ligand binding interactions at opposed G-tetrads. This technique revealed that the ligands TmPyP4, PhenDC3, and PDS have differential chemotype-specific binding preferences for individual G-tetrads of a model genomic G4 structure. ; This work was supported by the following grants: Cancer Research UK Programme, BBSRC BB/K018043/1 and EPSRC EP/ K039520/1. We thank Dr Chris Lowe for his constructive comments and for proofreading the manuscript.