The therapeutic potential of small molecules targeting G-quadruplexes has gained credibility since such structures were shown to form in human cells and to be highly prevalent in the human genome, most notably at telomere ends and in oncogene promoters. Herein, we perform whispering gallery mode (WGM) sensing for monitoring DNA–small molecule interactions. Unlike most existing technologies, WGM sensing offers numerous advantages including high sensitivity, real-time analysis, easy access to kinetic parameters, and much lower cost than current gold standards. In this work, interactions of five known DNA-binding ligands with either G-quadruplex or duplex DNA immobilized on a sphere microresonator have been assessed. The induced shift of the resonant mode from quadruplex (or duplex)–ligand binding was used to estimate kinetic parameters. Association and dissociation rate constants (kon and koff, respectively) as well as dissociation equilibrium constants (KD) were measured for these five ligands binding to both duplex and quadruplex DNA.