Purpose: The objective of this study was to evaluate and compare the accuracy and image quality of root surface area (RSA) measurements obtained with various cone-beam computed tomography (CBCT) protocols, relative to the gold standard of micro-computed tomography (CT), in an in vitro setting. Materials and Methods: Four dry human skulls were scanned using 8 different protocols, with voxel sizes of 0.15 mm, 0.3 mm, and 0.4 mm. Three-dimensional models of the selected teeth were constructed using CBCT and microCT protocols, and the RSA was automatically measured by the image-processing software. The absolute difference in the percentage of the RSA (%ΔRSA) was calculated and compared across the 8 CBCT protocols using repeatedmeasures analysis of variance. Finally, image quality scores of the RSA measurements were computed and reported in terms of percent distribution. Results: No significant differences were observed in the %ΔRSA across the 8 protocols (P>0.05). The deviation in %ΔRSA ranged from 1.51% to 4.30%, with an increase corresponding to voxel size. As the voxel size increased, the image quality deteriorated. This decline in quality was particularly noticeable at the apical level of the root, where the distribution of poorer scores was most concentrated. Conclusion Relative to CBCT protocols with voxel sizes of 0.15 mm and 0.3 mm, the protocols with a voxel size of 0.4 mm demonstrated inferior image quality at the apical levels. In spite of this, no significant discrepancies were observed in RSA measurements across the different CBCT protocols.