The aim of the present study was to compare radiographic assessment of interproximal bone loss using a loupe with a 0.1 mm calibrated grid and a computer-assisted analysis system (LMSRT). In 35 patients suffering from untreated advanced periodontal disease, 62 standardized radiographs were taken presurgically. The horizontal and vertical angulation difference of the central beam from the orthoradial projection was calculated for each radiograph. At the time of surgery, for 115 interproximal defects, the distances from the cementoenamel junction (CEJ) to alveolar crest (AC), and CEJ to bottom of the bony defect (BD) were measured. In all radiographs, the linear distances CEJ to AC, and CEJ to BD were assessed using a loupe and LMSRT. Comparison between radiographic and intrasurgical assessments was performed using paired t-tests. A stepwise multiple linear regression analysis was used to evaluate factors that influence the discrepancy between radiographic and intrasurgical measurements. Both analyzing techniques underestimated interproximal bone loss as compared with intrasurgical measurements (CEJ-AC: loupe: 0.86 +/- 1.84 mm [p < 0.001]; LMSRT: 0.58 +/- 1.86 mm [p < 0.005]; CEJ-BD: loupe: 1.22 +/- 2.33 mm [p < 0.001]; LMSRT: 0.80 +/- 2.09 mm [p < 0.001]). LMSRT underestimated interproximal bone loss significantly less than the loupe (p < 0.001). The difference between LMSRT and intrasurgical assessments was modulated by the factors of vertical and horizontal angulation difference and defect depth (p < 0.1). Orthoradial projection reduced underestimation of radiographic assessment of bone loss. LMSRT underestimated interproximal bone loss to a lesser extent than conventional evaluation by loupe.