Background: Chest X-ray is one of the examinations required for an annual health checkup. The interaction of radiation to the medium produces free radicals, which consequently causes biological changes either structural or properties of the cells. Whether the radiation from Chest X-ray upright technique affects the plasma membrane fluidity of thalassemic red blood cells (RBCs) is still unclear. Materials and Methods: Whole blood samples of non-thalassemia group (G1), non-clinically significant thalassemia group (G2) and clinically significant thalassemia group (G3) were irradiated with the Chest X-ray upright technique with a dose of 6.57 µSv (90 kVp). One-tube osmotic fragility (OF) test, dichlorophenol-indophenol (DCIP) test for hemoglobin E (HbE) screening and glucose-6-phosphate dehydrogenase (G6PD) activity analysis were performed on those samples. Results: The % OF values of G1 and G3, between irradiated and non-irradiated RBCs, were comparable, while those of G2 were significantly increased. However, the irradiation did not affect HbE screening test by DCIP technique in G2. In addition, it did not alter the G6PD activity in both thalassemia and non-thalassemia samples.  Conclusion: Chest X-ray upright technique with a dose of 6.57 µSv (90 kVp) did not alter the membrane fluidity and the G6PD activity of thalassemic and non-thalassemic RBCs. Thus, it did not affect the thalassemia screening by OF and DCIP tests. However, increasing % OF value in HbE trait might be used as one of the screening test for HbE trait.