Currently, small molecules able to interact specifically with G-quadruplex DNA (G-quadruplex ligands) are intensively studied since they appear to inhibit the growth of cancer cells via an unprecedented mode of action based on structural perturbation of telomeres. It is thus of crucial interest to develop methods that enable easy, rapid and reliable detection of valuable candidates. Herein, we would like to report on the G4-FID assay, a simple fluorescence screening method based on the displacement of a DNA light-up probe (thiazole orange) from both quadruplex and duplex DNA, therefore allowing a concomitant evaluation of the quadruplex-affinity and quadruplex- over duplex-selectivity of the tested candidates.