High-mannose-type glycans on glycoproteins play pivotal roles in protein folding, secretion, and degradation in the endoplasmic reticulum (ER). Chemical approaches using structurally defined synthetic glycan probes and inhibitors have enabled the precise elucidation of the functions of glycan-related proteins involved in ER glycoprotein quality control (ERQC), including UGGT1, glucosidase II, calreticulin (CRT), and EDEM family proteins. This chapter highlights how synthetic high-mannose-type glycans have revealed the molecular-level functions of these proteins, including substrate recognition dependent on the branching patterns and aglycone hydrophobicity. Our study demonstrated that UGGT1 preferentially glucosylates glycan substrates with hydrophobic aglycones and recognizes specific glycan branching patterns, whereas glucosidase II exhibits distinct reactivities toward glycan isomers. Moreover, synthetic trisaccharides clarified EDEM’s preference for branched chains, and selective inhibitors elucidated discrete mannose-trimming pathways leading to the production of secretion or degradation signals. The reconstructed glycan profile method further revealed disease-related alterations in ER glycan processing. These findings underscore the power of chemical glycan probes in dissecting the mechanisms of ERQC at the molecular level and provide insights into the relationship between glycan structures and protein folding states, advancing our understanding of ER-associated diseases.