Intracellular amine metabolite changes were quantified from hyperglycemic human aortic endothelial cells (HAECs) as a model for macrovascular complications of diabetes. Amines were selectively tagged using the N-hydroxysuccinimide ester (NHS) based isobaric tag DiART (Deuterium isobaric Amine Reactive Tag), synthesized in house. DiART labeling improved chromatographic resolution of derivatized amines, resulted in 100-fold signal-to-noise enhancement in mass spectrometry (MS) analyses, and allowed multiplex quantification of four samples concurrently through tandem MS fragmentation. Targeted measurement of 31 DiART-tagged amines demonstrated the limits of detection below 10 nM/100 amol and averaged RSDs less than 5%. Examination of endothelial cells exposed to short-term hyperglycemia resulted in significant changes to alanine, proline, glycine, serine, and glutamine compared to osmotic controls. Discovery of proline elevation in hyperglycemic endothelial cells suggests a role of proline in hyperglycemia-mediated oxidative stress. Exposure of endothelial cells to high glucose for 7 days resulted in reduced cell number and significant changes to 21 amines relative to cell number. Prominent amine elevation from long-term hyperglycemia include aminoadipate as a sign of lysine breakdown through oxidative stress; cystathionine, hypotaurine, and proline indicating an antioxidant response; and glutamine/glutamate as substrate level activators of additional metabolic pathways. This report is the first investigation of amine changes to hyperglycemic endothelial cells and offers new insights into the pathophysiology of diabetic complications.