Added PubMed Id Protein glycosylation is an essential posttranslational modification in all domains of life. Its impairment in human can result in severe diseases named Congenital Disorders of Glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation, O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, or ALG11 genes revealed substantial reduction in the levels of corresponding proteins. The abundance of the other GTs, however, remained unchanged on transcript as well as on protein level, indicating that there is no fail-safe mechanism for this essential pathway. The established MRM assay is easily shared with the scientific community via the open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps even while using different LC-MS hardware.