Therapeutic Fc-fusion proteins, created by linking bioactive peptides or receptor proteins to the Fc moiety of IgG, are currently being developed. In this development process, a Gly-Gly-Gly-Ser linker (G4S linker) is often used to link the peptide/protein and the Fc portion. O-xylose-type core glycans of glycosaminoglycan are known to attach to the Ser residue on the GSG motif in the G4S linker peptide repeats of the Fc fusion protein produced using the Chinese hamster ovary (CHO) cell expression system. In addition, a recent report demonstrated that unexpected mucin-type O-glycosylations occurred on a peptide in a bioactive peptide-Fc fusion protein; this glycosylation affected the bioactivity of the peptide. Therapeutic proteins with non-natural structures, such as Fc-fusion proteins, undergo unintended O-glycosylations; therefore, it is increasingly important to conduct detailed O-glycosylation analysis of fusion proteins during the developmental stages. In this paper, we have summarized recent reports on the unexpected O-glycosylation in fusion proteins, general O-glycosylation types and sequence motifs, and O-glycosylation analytical techniques involving O-linked oligosaccharide analysis and site-specific O-glycosylation analysis using LC/MS. In addition, we have introduced site-specific O-glycosylation analysis of Fc-fusion proteins with GS linker peptides by LC/MS using higher-energy collisional dissociation-tandem mass spectrometry (HCD-MS/MS) and electron-transfer dissociation (ETD)-MS/MS to obtain preferential dissociation of the peptide moiety in the glycopeptide.