Background: People occasionally undergo medical diagnostic X-ray examinations and expose their red blood cells to radiation. Radiation that is generated from medical diagnostic X-ray machines is widely used in medical diagnoses. One of the important parameters is kilo-voltage (kV) that is applied across the X-ray tube in medical diagnostic X-ray machines. Kilo-voltage influences the radiation dosage. The aim of this study is to determine the hemolysis, osmotic fragility, and fluorescence anisotropy value on irradiated red blood cells as a function of kV during medical diagnostic X-ray examinations.  Materials and Methods: The kV, kilo-voltage that is applied across an  X-ray tube, of a medical diagnostic X-ray machine was operated at 50, 70 and 100 kV. We determined the hemolysis, osmotic fragility, and fluorescence anisotropy value in red blood cells at 0.5 and 4 hours post-irradiation. In order to determine hemolysis and osmotic fragility, the release of hemoglobin was measured by spectrophotometry technique. 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as a molecular probe for determining fluorescence anisotropy value by fluorescence anisotropy technique. Non-irradiated red blood cells served as the control. Results: For the 50, 70, and 100 kV of medical diagnostic X-rays, the hemolysis, osmotic fragility, and fluorescence anisotropy values of irradiated red blood cells at 0.5 and 4 hours post-irradiation did not significantly change when compared to the control.  Conclusion: Our results suggested that 50, 70, and 100 kV of medical diagnostic X-ray did not influence hemolysis, osmotic fragility, and fluorescence anisotropy values of irradiated red blood cells.