Among post-translational modifications, non-enzymatic glycation is of particular relevance, since it plays a pivotal role in the development of long-term diabetic complications. Beside continuous advances in treatment of those deleterious effects, aspirin was shown to prevent proteins from excessive glycation. However, the interplay between aspirin-induced acetylation and protein glycation was poorly investigated. In this project we studied an in vitro model of the influence between high glucose and aspirin effect in the main blood compartments using a label-free mass spectrometry approach. We showed that aspirin reduces protein glycation in the major plasma proteins, different haemoglobin subunits and proteins involved in platelet activation and inflammation. Furthermore, we determined the impact of hyperglycaemia on aspirin-induced acetylation of platelets proteins from diabetic patients, focusing our attention on COX-1. We demonstrated that diabetes is associated with an impaired aspirin effectiveness, providing a potential mechanism to the platelet hyper-reactivity observed in aspirin-treated diabetic patients.