Long-term hyperglycemia in diabetic patients leads to human serum albumin (HSA) glycation, which may impair HSA function as a transport protein and affect the therapeutic efficacy of anticoagulants in diabetic patients. In this study, a novel mass spectrometry (MS) approach was developed to reveal the differences in the profiles of HSA glycation sites between diabetic and healthy subjects. K199 was the glycation site most significantly changed in diabetic patients, contributing to different interactions of glycated HSA (gHSA) and normal HSA with two types of anticoagulant drugs, heparin and warfarin.