UGGT1 is called as a folding sensor protein that recognizes misfolded glycoproteins and selectively glucosylates high-mannose-type glycans on the proteins. However, conventional approaches using naturally occurring glycoproteins is not optimum in performing precise analysis of the unique properties of UGGT1. We have demonstrated that high-mannose-type glycans, in which various hydrophobic aglycons were introduced, act as good substrates for UGGT1 and are useful analytical tools for its characterization. Moreover, we found that UGGT2, an isoform UGGT1, is also capable of glucosylating these synthetic substrates. Our strategy stemmed on synthetic chemistry has been further strengthened by total synthesis of homogeneous glycoproteins in correctly folded as well as in intentionally misfolded forms.