Visualization of guanine-rich oligonucleotides that fold into G-quadruplex (G4) helical structures is of great interest in cell biology. There is a large body of evidence that suggests that these noncanonical structures form in vivo and play important biological roles. A promising recent development highlighted fluorescence lifetime imaging microscopy (FLIM) as a robust technique for the direct and quantitative imaging of G4s in live cells. However, this method requires specialized, bespoke synthetic dyes that are not widely available. Herein, we demonstrate that the fluorescence lifetime of commercially available environmentally sensitive dyes Thioflavin T (ThT) and Thiazole Orange (TO) is strongly dependent on the type of DNA topology they bind to, with G4s showing long and distinctive decay times that should allow G4 detection in the biological environment. We applied this observation to visualize G4s in live U2OS cells using FLIM of ThT, upon alteration in G4 levels due to competitive binding or nuclease treatment of cells.