A curated library of circular dichroism spectra of 23 G-quadruplexes of known structure was built and analyzed. The goal of this study was to use this reference library to develop an algorithm to derive quantitative estimates of the secondary structure content of quadruplexes from their experimental CD spectra. Principal component analysis and singular value decomposition were used to characterize the reference spectral library. CD spectra were successfully fit to obtain estimates of the amounts of base steps in anti-anti, syn-anti or anti-syn conformations, in diagonal or lateral loops, or in other conformations. The results show that CD spectra of nucleic acids can be analyzed to obtain quantitative structural information about secondary structure content in an analogous way to methods used to analyze protein CD spectra.