G-quadruplexes are noncanonical secondary structures formed in DNA sequences containing consecutive runs of guanines. It has been shown that the 3′ G-rich single-stranded overhangs of human telomeres can form G-quadruplex structures, and the human telomeric DNA G-quadruplexes are considered attractive targets for anticancer drugs. G-quadruplex-interactive compounds have been shown to inhibit telomerase access as well as telomere capping. Nuclear magnetic resonance (NMR) spectroscopy is a powerful method in determining the G-quadruplex structures under physiologically relevant conditions. We present the NMR and biophysical methodology used in our research group for the study of G-quadruplex structures in physiologically relevant solution and their interactions with small-molecule compounds.