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Background and Objectives: Beta-thalassemia continues to be a cause of significant burden to the society, particularly in the poorer developing countries. The objective of the present study was to evaluate the validity of “NESTROFT” (Naked Eye Single Tube Red Cell Osmotic Fragility Test) as a useful screening tool in the diagnosis of beta thalassemia trait. Material and Methods: The present study was conducted on 150 subjects in the department of haematology in a tertiary health care center in north Indian state of Punjab. In Group I, 111 cases diagnosed as microcytic hypochromic anaemia were selected. In Group II, 39 individuals (the family members of known cases of beta thalassemia major) were selected. Complete haemogram, NESTROFT and HbA2 levels by electrophoresis were done and the results were tabulated and analyzed statistically. Results: Of the 111 cases in Group I, 20 (18%) gave positive results with NESTROFT while 91 cases (82%) tested negative. In Group II, out of 39 cases, 30 (76.92%) tested positive with NESTROFT while 9 gave a negative result. In Group I, out of 20 NESTROFT positive cases, only 3 had HbA2 levels more than 3.5%. In Group II, all the 30 NESTROFT positive cases had HbA2 levels more than 3.5%. The test showed a sensitivity of 100%, specificity of 85.47%, a positive predictive value of 66% and a negative predictive value of 100%. Conclusion: Thus, NESTROFT is a valuable, cost-effective screening test for beta thalassemia trait and appears to be a valid test in rural setting with financial constraints.