Introduction: Early diagnosis and therapeutic intervention can improve the outcome of acute myocardial infarction (AMI). However, there are no satisfactory cardiac biomarkers for the diagnosis of AMI within 6 hours of onset of symptoms. Among the novel biomarkers of AMI, we tested the diagnostic accuracy of Heart type Fatty Acid Binding Protein (HFABP) in prospectively recruited patients with independently adjudicated outcomes. Methods: Prospective observational study was conducted at the Department of Pathology and Department of Cardiology, Dr Ram Manohar Lohia Institute of Medical Sciences Lucknow. After taking informed consent, 80 cases of chest pain suggestive of coronary origin (within 6 hours) were included. Results: Group a included 55 patients while Group B included 25 patients. In our study on Receiver operator curve (ROC) analysis, the AUC (Area under curve) was 0.846 (95% C.I. 0.743 to 0.948; p value <0.001) for HFABP. The optimum cut-off value for HFABP to diagnose AMI was 6.38 ng/ml with a Sensitivity, Specificity, PPV (Positive Predictive Value) and NPV of 86.4%, 84.5%, 67.9% and 94.2% respectively. Conclusion: The optimum cut-off value for HFABP to diagnose AMI was found to be 6.38 ng/ml were in concordance with similar studies. A high sensitivity and NPV is essential for the early ‘rule out’ of AMI patients. Since more than 70% of patients who present with acute chest pain to an emergency department do not have AMI, HFABP can be used as rule out tool to prevent unnecessary admissions of patients suspected for AMI considering its high sensitivity and NPV. Though, it was not a reliable marker for AMI diagnosis because of low specificity and positive predictive value. Further larger studies are needed to understand whether HFABP can add incremental value in rule-in AMI.