Secondary structure of the glycosylphosphatidylinositol attachment signal (GPI-AS) in GPI-anchored proteins, usually cleaved from the mature protein in eukaryotic cells, was evaluated using circular dichroism (CD). GPI-AS was expressed in Escherichia coli and was purified as a green fluorescent protein (GFP)-fused recombinant protein. The secondary structure was monitored by observing the far-UV CD bands. The α-helix content increased by 3.7% (indicates 13 amino acids) in GFP-fused GPI-AS, indicating that GPI-AS tends to have an α-helical structure. The method we propose in this study can be used to evaluate the secondary structure of the signal regions, which are absent in the mammalian mature proteins.