The aim of this study was to develop a quantitative radiographic method for assessment of experimental osteoporosis in Leghorn breed laying hens. Three groups of 24 Leghorn pullets were reared in cage and fed a ration containing different calcium levels, including 3.55, 2.075 and 0.6 percent (for groups 1-3, respectively). The hens were fed this diet from 17 weeks of age to the end of the experiment. At 20, 28 and 36 weeks of age, 8 hens from each group were selected randomly. Radiographs were obtained from the tibiotarsus and the humerus of each hen. Radiographs were digitized using a camera and assessed by “Image J” software. Bone radiopacities and bone cortex/diameter (C/D) ratios were measured. The hens were sacrificed and the bone ash and calcium contents were measured. Bone densities of the birds in different groups were significantly different at just 36 weeks of age; they were greater in the tibiotarsus bone of the control group than in the tibiotarsus bone of median (P=0.02) and with the low calcium (P=0.007) groups. Humerus densities were also greater in control group compared with that of median (P=0.04) and with low calcium (P=0.0004) diet group. Cortex/diameter index of the tibiotarsus bone was different in all three stages between control and the two other groups, while there were no significant differences between the humeri C/D indices and the three groups in the first stage. Humeri C/D indices of the second and third stages had significant differences between control and the two other groups (P≤0.05). This study showed that radiographic evaluation of bone density is valuable just in progressed osteoporosis, while C/D index can be used for diagnosis of osteoprotic bones in earlier stages