N-glycosylation mediates many biological functions. Genetic defects in the N-glycosylation pathway cause >35 inherited human disorders called congenital disorders of glycosylation (CDGs). As a result, some N-glycosylation sites are unoccupied. Serum transferrin is a diagnostic marker for these patients, but there are no corresponding cellular markers to assess glycosylation competence. Therefore, we engineered a green fluorescent protein (GFP) construct to measure N-glycosylation site occupancy. We designed an endoplasmic reticulum-retained GFP biomarker whose fluorescence is lost when it is N-glycosylated due to steric hindrance by the glycan. This marker is a highly sensitive indicator of N-glycosylation site occupancy. In CDG cells carrying the GFP construct, a 25% decrease of glycosylation efficiency induces a 5-fold increase in fluorescence, while cDNA complementation of the genetic defect results in a 5-fold decrease in fluorescence. This engineered GFP detects impaired N-glycosylation in multiple cell lines, including CHO cells, HeLa cells, normal and patient fibroblasts, induced pluripotent stem cells (iPSCs), and human embryonic stem cells (hESCs). This marker is a highly sensitive tool to study N-glycosylation site occupancy. It can be used to screen for compounds that reverse poor N-glycosylation site occupancy.