Abstract: A sulfonyl chloride-labeled glycopeptide standard Dabsyl-Gly-Glu-Asn-(GlcNAc4Man3)-Arg was used as the initial substrate to study the minimal sugar substrate of N-Glycosidase PNGase H⁺. Enzymatical method was used to obtain target substrates by the treatment with β-N-acetyl-hexosaminidase,α-mannosidase,β-mannosidase and β4GalT1. Five glycopeptide substrates were synthesized in total. They were applied in the determination of minimal sugar substrate of N-glycosidase PNGase H⁺. High performance liquid chromatography was used to detect the enzymatic hydrolysis of PNGase H⁺ on these five glycopeptide substrates. It was found that five glycopeptide substrates were successfully obtained,including Dabsyl-Gly-Glu-Asn-(GlcNAc2Man3)-Arg,Dabsyl-Gly-Glu-Asn-(GlcNAc2Man)-Arg,Dabsyl-Gly-Glu-Asn-(GlcNAc2)-Arg,Dabsyl-Gly-Glu-Asn-(GlcNAc)-Arg and Dabsyl-Gly-Glu-Asn-(GlcNAc2Gal)-Arg with concentration of 8.3,7.0,6.5,4.5,6.5 pmol/μL,respectively. And PNGase H⁺ could completely release sugar from four different glycopeptides with 5,3 and 2 sugar unit,while it had almost no effect on glycopeptide with a single GlcNAc. Therefore,it was concluded that the minimal sugar substrate of PNGase H⁺ should be glycopeptide with N-acetylchitobiose.