We hypothesize that inclusion of denatured proteins in the set of reference native proteins may better represent the unordered form in the current circular dichroism (CD) analyses of proteins involving unfolding ones. Adding three denatured-protein spectra and one oligopeptide spectrum to 16 reference protein spectra markedly improved the correlation coefficients (r) between CD calculations and X-ray determinations for the unordered form and, to a lesser extent, for β-turn, but the r-values for α-helix and β-sheet decreased slightly. With 20 reference proteins the estimates of the unordered form of denatured proteins were significantly improved. Thus, we suggest that as a compromise the new set of reference proteins be used for estimating the changes in conformation for unfolding proteins. However, the current use of 16 reference native proteins appears to be adequate for CD analysis of native proteins and the expansion to 20 reference proteins including denatured ones may not enhance the analysis of native proteins.