Eight [Ru(bpy)₂ L]²⁺ and three [Ru(phen)₂ L]²⁺ complexes (where bpy = 2,2'-bipyridine and phen = 1,10-phenanthroline are ancillary ligands, and L = a polypyridyl experimental ligand) were investigated for their G-quadruplex binding abilities. Fluorescence resonance energy transfer melting assays were used to screen these complexes for their ability to selectively stabilize human telomeric DNA variant, Tel22. The best G-quadruplex stabilizers were further characterized for their binding properties (binding constant and stoichiometry) using UV-vis, fluorescence spectroscopy, and mass spectrometry. The ligands' ability to alter the structure of Tel22 was determined via circular dichroism and PAGE studies. We identified me₂ allox as the experimental ligand capable of conferring excellent stabilizing ability and good selectivity to polypyridyl Ru(II) complexes. Replacing bpy by phen did not significantly impact interactions with Tel22, suggesting that binding involves mostly the experimental ligand. However, using a particular ancillary ligand can help fine-tune G-quadruplex-binding properties of Ru(II) complexes. Finally, the fluorescence "light switch" behavior of all Ru(II) complexes in the presence of Tel22 G-quadruplex was explored. All Ru(II) complexes displayed "light switch" properties, especially [Ru(bpy)₂ (diamino)]²⁺ , [Ru(bpy)₂ (dppz)]²⁺ , and [Ru(bpy)₂ (aap)]²⁺ . Current work sheds light on how Ru(II) polypyridyl complexes interact with human telomeric DNA with possible application in cancer therapy or G-quadruplex sensing.