G-quadruplexes are noncanonical, four-stranded nucleic acid secondary structures formed in sequences containing consecutive runs of guanines. These G-quadruplex structures have been found to form in nucleic acid regions of biological significance, including human telomeres, gene promoters, and untranslated regions of mRNA. Thus, they are considered attractive therapeutic targets. Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for understanding the structures of G-quadruplexes and their interactions with small molecules under physiologically relevant conditions. Here, we present the NMR methodology used in our research group for the study of DNA G-quadruplex structures in physiologically relevant solution and their ligand interactions.