Guided Bone Regeneration is a common procedure, yet, as new grafting materials are being introduced into the market, a reliable evaluation method is required. Critical size defect in animal models provides a valid simulation, followed by histological sections to evaluate bone formation. However, histology is destructive, two dimensional and technique-sensitive. In this study we developed a novel volumetric Micro-CT analysis to quantify new bone formation characteristics. Eight adult female New Zealand white rabbits were subjected to calvarial critical size defects. Four circular defects, 8-mm in diameter were preformed in each animal, to allow random allocation of four treatment modalities. All calvarias were scanned using Micro-CT. Each defect was segmented into four equal parts: pristine bone, outer, middle, and inner. Amira software was used to calculate the percentage of new bone volume in each region and compare it to that of the pristine bone. All grafting materials demonstrated that new bone formation decreased as it moved inward. Only the inner region differed across grafting materials (p=0.001). The new Micro-CT analysis allowed us to divide each defect into 3D regions providing better understanding of the bone formation process. Amongst the various advantages of the Micro-CT, it enables us to independently quantify the filler materials and the newly formed bone, and to describe the defect morphology in 3D (bi- vs. uni-cortical defects). Micro-CT will probably become a standard tool for animal model research in the future, owing to the quantitative 3D data and accuracy that it provides. As new materials are introduced into dental practice, new quantitative methods are required to evaluate grafting material properties. Providing an insight into the inner region of the defect can better predict the regenerative potential of the bone replacement filler, therefore, the suggested Micro-CT analysis is beneficial for further development of clinical approaches.