Updated project metadata. Protein glycation is a highly disease-relevant concentration-dependent non-enzymatic reaction of reducing sugars with amine groups of proteins to form early as well as advanced glycation products (AGEs). To complement our blood proteomics studies in diabetics, we here established higher-energy collisional dissociation (HCD) fragmentation on Orbitrap mass spectrometers for analyzing protein glycation. We evaluated parameters to most efficiently fragment and identify early glycation products on in-vitro glycated model proteins. We then applied our optimized workflow for glycation analysis of the entire HeLa proteome as well as for single-run analysis of undepleted and unenriched blood plasma and whole blood. We conclude that HCD fragmentation is well suited for analyzing glycated peptides when integrating the dominant neutral loss into the analysis and that single-run plasma proteomics measurements have great potential to diagnosing the diabetic status of patients.