Background and objectives: Creatine phosphokinase-myocardial band fraction (CPK-MB) and cardiac troponin I (cTnI) are cardiac specific biochemical markers which are raised in myocardial ischemia. The aim of this study was to determine cardiac injury by comparing the levels of cardiac enzymes CPK-MB and cTnI in atrial septal defect (ASD) patients whose operative repair was done under cardiopulmonary bypass (CPB) using beating heart technique with and without the application of aortic cross clamp. Materials and Methods: This study was carried out in the Department of Cardiothoracic and Vascular Surgery in a Tertiary Care Hospital over a period of 2 years. A total of 60 atrial septal defect (ASD) patients were operated and repair of the defect was done under the CPB using beating heart technique. Aortic cross clamp was applied in 22 patients (Group-A) while 38 patients were operated without cross clamp (Group-B) during the procedure. Blood samples were collected 24 hours prior and 12 hours post procedure for the estimation of CPK MB and cTnI levels. Results: Mean age of the atrial septal defect patients was 23.83±10.97 years and 60% and 40% of the patients were females and children (age < 18 years) respectively. Serum CPK-MB and cTnI l levels were in the normal range in all the patients before surgery and increased significantly post procedure. Twelve hours after surgery, the mean CPK-MB and cTnI levels were significantly low in Group-B patients compared to Group-A patients (CPK-MB: 56.39±23.55 U/L vs. 34.38±15.97U/L , p= 0.0004; cTnI: 9.37±4.97 ng/ml vs. 5.92±4.17ng/ml, p = 0.009). Conclusion: Post surgery CPK-MB and cTnI levels were significantly higher in ASD patients who underwent CPB surgery with aortic cross clamp compared to those in whom aortic cross clamp was not applied. Therefore, application of aortic cross clamp during the procedure induces greater levels of ischemic injury to the heart. IMC J Med Sci. 2023; 17(1): 001. DOI: https://doi.org/10.55010/imcjms.17.001 *Correspondence: Feroze Mohammad Ganai, Department of CVTS, Superspeciality Hospital, Shireen Bagh, Srinagar, Jammu and Kashmir, India. Email address: ferose999@yahoo.com