Circular dichroism (CD) is a phenomenon that arises from the differential absorption of left- and right-handed circularly polarized light, and may be seen with optically active molecules. CD spectroscopy provides useful spectral signatures for biological macromolecules in solution, and provides low-resolution structural information about macromolecular conformation. CD spectroscopy is particularly useful for monitoring conformational changes in macromolecules upon environmental perturbations. G-quadruplex structures show unique CD spectral signatures, and CD is an important tool for characterizing their formation and global structure. This protocol offers step-by-step methods for determining reliable and reproducible CD spectra of quadruplex structures and normalizing the spectra for presentation. CD spectra properly normalized with respect to quadruplex concentration and path length are required to facilitate accurate comparison of results among laboratories. The standard operating procedures proposed are recommended to make such comparison accurate and informative. © 2017 by John Wiley & Sons, Inc.