Protein engineering is a powerful tool for designing or modifying therapeutic proteins for enhanced efficacy, greater safety, reduced immunogenicity, and better delivery. GGGGS [(G4S)n] linkers are commonly used when engineering a protein, because of their flexibility and resistance to proteases. However, post-translational modifications (PTMs) can occur at the Ser residue in these linkers. Here, we report, for the first time, the occurrence of O-xylosylation at the serine residue in (G4S)n>2 linkers. The O-xylosylation was discovered as a result of molecular mass determination, peptide mapping analysis, and MS/MS sequencing. Our investigation showed that (i) O-xylosylation is a common PTM for (G4S)(n>2) linkers; (ii) GSG is the motif for O-xylosylation; and (iii) the total amount of xylosylation per linker increases as the number of GSG motifs in the linker increases. Our investigation has also shown that the O-xylosylation level is clone-dependent, to a certain degree, but the xylosylation level varies considerably among the proteins examined-from <2% to >25% per linker-likely depending on the accessibility to the sites by the xylosyltransferase. Our work demonstrates that potential therapeutic proteins containing (G4S)n linkers should be closely monitored for O-xylosylation in order to ensure that drugs are homogeneous and of high quality. The strategies for elimination and reduction of O-xylosylation were also examined and are discussed.