G-quadruplexes (G4s) have become one of the most exciting nucleic acid secondary structures. A noncanonical, four-stranded structure formed in guanine-rich DNA and RNA sequences, G-quadruplexes can readily form under physiologically relevant conditions and are globularly folded structures. DNA is widely recognized as a double-helical structure essential in genetic information storage. However, only ~3% of the human genome is expressed in protein; RNA and DNA may form noncanonical secondary structures that are functionally important. G-quadruplexes are one such example which have gained considerable attention for their formation and regulatory roles in biologically significant regions, such as human telomeres, oncogene-promoter regions, replication initiation sites, and 5′- and 3′-untranslated region (UTR) of mRNA. They are shown to be a regulatory motif in a number of critical cellular processes including gene transcription, translation, replication, and genomic stability. G-quadruplexes are also found in nonhuman genomes, particularly those of human pathogens. Therefore, G-quadruplexes have emerged as a new class of molecular targets for drug development. In addition, there is considerable interest in the use of G-quadruplexes for biomaterials, biosensors, and biocatalysts. The First International Meeting on Quadruplex DNA was held in 2007, and the G-quadruplex field has been growing dramatically over the last decade. The methods used to study G-quadruplexes have been essential to the rapid progress in our understanding of this exciting nucleic acid secondary structure.