Human thyroglobulin (Tg), which has many glycosylation sites, is an essential protein produced by the human thyroid glands. Although human Tg N-glycans play critical roles in the cellular events of the Thyroid gland, the site-specific distribution of glycan structures has not been studied in detail. This study aimed to profile human Tg N-glycosylation sites and their glycan contents by using high-throughput glyco-analytical strategies, including glycopeptide and glycan levels. The sulfated complex and hybrid type N-glycan species were determined by the analysis of the human Tg samples with HPLC-HILIC-FLD-MS/MS. It was found that all fucosylated N-glycans carried fucose residue on their N-glycan core structure. The human Tg was digested with multiple enzymes by applying both in-gel and in-solution protocols to enhance site-specific glycosylation analysis. In total, 17 out of 20 N-glycosylation sites were characterized. It was noticed that 6 N-glycosylation sites contain only high-mannose type glycans, while other regions include complex and hybrid type glycans. In addition, sulfated gly-coform structures were detected at the glycopeptide level in glycosylation sites containing complex and hybrid type glycans. It is expected that the results obtained from this study will contribute to functional studies to be conducted on human Tg protein.Biological significance: N-glycans of human thyroglobulin modulate thyroid hormone synthesis both in vivo and in vitro. Therefore, a comprehensive analysis of the N-glycosylation sites of human thyroglobulin is essential to improve our understanding of the function of its N-glycans. The present research significantly expanded the knowledge regarding N-glycosylation profiles of human thyroid thyroglobulin protein. For instance, as high-lighted here, sulfated N-glycan structures were characterized using comprehensive glyco-analytical strategies. N-glycan patterns for the sites Asn110, Asn1869, and Asn2122 were described for the first time in this current work. In addition, N-glycan structures containing core-fucosylation and bisecting types were confirmed for all determined glycosylation sites.