AbstractG‐quadruplexes (G4s) are a kind of non‐canonical nucleic acid secondary structures, which involve in various biological processes in living cells. The relationships between G4s and human diseases, such as tumors, neurodegenerative diseases, and viral infections, have attracted great attention in the last decade. G4s are considered as a promising new target for disease treatment. For instance, G4 ligands are reported to be potentially effective in SARS‐COV‐2 treatment. However, because of the lack of analytical methods with high performance for the identification of intracellular G4s, the detailed mechanisms of the biofunctions of G4s remain elusive. Meanwhile, through demonstrating the principles of how the G4s systematically modulate the cellular processes with advanced detection methods, biochemical targeting of G4s in living cells can be realized by chemical and biological tools and becomes useful in biomedicine. This review highlights recent methodological advances about intracellular G4s and provides an outlook on the improvement of the bioanalysis and biochemical targeting tools of G4s.