Objective: To evaluate the utility of NESTROFT as a screening test for carriers of thalassemia major. Design: prospective study. Settings and participants: Total 121 clinically normal siblings, of thalassemia major patients who are registered at thalassemia welfare society of J K LON hospital Kota, were selected randomly for study. Intervention: All cases were investigated for CBC, Mentzer's index, NESTROFT and HbA2 estimation by HPLC method. Result: Total 121 cases were screened for thalassemia carrier by HbA2 estimation using HPLC technique. Out of which total 59 (48.76%) cases showed HbA2 level ≥3.5%considered as carrier while rest 62 (51.24%) cases were having normal Hemoglobin pattern. The Sensitivity, specificity, positive predictive value and negative predictive value of MCV<80 are 89.83%, 46.77%, 61.62% and 82.85% respectively. The Sensitivity, specificity, positive predictive value and negative predictive value of Mentzer's index are 83.05%, 97.36%, 96.07% and 88.09 % respectively. The Sensitivity, specificity, positive predictive value and negative predictive value of NESTROF test are 93.22%, 88.70%, 88.7% and 93.22 % respectively. So NESTROFT is better test (sensitivity is 93.22% and negative predictive value is 93.22%) for screening for Thalassemia carrier. Conclusion: NESTROF test is reliable, cost effective and better screening test for Thalassemia carrier detection. Positive cases can be confirmed by HPLC. NESTROFT can be used in mass hemoglobinopathies screening programmes.