Polyprenol reductase is an enzyme encoded by the SRD5A3 gene, which catalyzes the synthesis of dolichol from polyprenols. Dolichol serves as a carrier for glycan precursors in N-linked glycosylation or monosaccharides in O-glycosylation, C-mannosylation, and GPI anchor biosynthesis. Pathogenic variants in SRD5A3 can result in a congenital disorder of glycosylation (CDG), SRD5A3-CDG, which is inherited in an autosomal recessive manner. Most serum proteins undergo glycosylation and changes in the glycosylation levels of numerous serum glycoproteins have been associated with pathological consequences. Despite the critical role of SRD5A3 in glycosylation, the impact of its deficiency on the glycosylation of serum proteins remains largely unexplored. In this study, we used tandem mass tags (TMT)-based multiplexed quantitation approach to analyze serum N-glycoproteins and proteins in SRD5A3-CDG patients and controls. We quantified 2,200 serum N-glycopeptides from 359 N-glycosites from 204 serum proteins. Extensive hypoglycosylation of serum proteins was observed in patients, with 245 of 291 dysregulated glycopeptides showing hypoglycosylation. These significantly changing glycopeptides belonged to several known abundant serum glycoproteins including haptoglobin, plasma serine protease inhibitor, alpha-1-B glycoprotein, alpha-2-macroglobulin and ceruloplasmin. We also detected changes in glycopeptides from albumin which has recently been shown to be glycosylated. Overall, our study provides novel insights into alterations in the glycosylation status of serum proteins in SRD5A3-CDG that could facilitate further investigations into diagnostics and therapeutics aspects of this CDG.