Purpose: the aim of this study was to evaluate the effect of different voxel sizes in volumetric analysis of prepared pulp space segmentation. Material and Methods: 20 teeth were imaged after endodontic preparation. Physical volume of prepared pulp spaces were used as a golden standard. The teeth were imaged by using CBCT at two different voxel sizes: (1) High definition mode (150μm) and (2) low dose mode (400μm). Materialise Mimics software and a semi-automated segmentation method were used to segment prepared pulp space. Results: the high definition mode (150μm) has acceptable agreement (73%) while the low dose mode (400μm) has unacceptable agreement (21%) for assessing the physical volume “gold standard”, and these differences are statistically significant. Conclusions: With increasing voxel sizes during scanning, there was decreasing in the accuracy of segmented volume measurements of maxillary centrals prepared pulp space.