A rapid differential diagnosis of the clinical conditions underlying chest pain is a relevant clinical issue. Specifically, a fast rule-in or -out of acute myocardial infarction (AMI) is mandatory to improve diagnostic outcome and cost-effectiveness of patient management. We demonstrated that Protein Kinase C (PKC) epsilon is selectively expressed by platelets from AMI patients, accounting for increased platelet activation. Thus, we hypothesized that PKCepsilon-expressing platelets may represent a pathophysiological marker of AMI that could be utilized in combination with troponin-I, the conventional marker of cardiac injury, to add diagnostic information in chest pain workup. In 94 chest pain patients consecutively admitted to Parma University Hospital, we tested the diagnostic performance of flow-cytometric detection of PKCepsilon expressing platelets in discriminating AMI vs. non-AMI conditions. We demonstrated that PKCepsilon-expressing platelets were significantly higher in patients with AMI. Flow cytometry detection of PKCepsilon-expressing platelets showed high sensitivity and specificity (87.5% and 84.4%, respectively) and good diagnostic accuracy (AUC: 0.875). The combination of PKCepsilon expressing platelets and cardiac troponin clearly discriminates patients with 100% and 0% of probability to be affected by AMI. Overall, we highlighted a dual marker strategy potentially useful for a rapid rule-in or -out of myocardial infarction in chest pain patients.