Subcritical calvarial defects are important to study bone regeneration during healing. In this study 1mm calvarial defects were created using trephine in the parietal bones of Sprague-Dawley rats (n=7) that served as in vivo defects. Subjects were sacrificed after 7 days and the additional defects were created on the harvested skull with the same method to serve as control defects. Raman spectroscopy is established to investigate mineral/matrix ratio, carbonate/phosphate ratio and crystallinity of three different surfaces; in vivo defects, control defects and normal surface. Results show 21% and 23% decrease in mineral/matrix after 7 days of healing from surface to in vivo and control to in vivo defects, respectively. Carbonate to phosphate ratio was found to be increased by 39% while crystallinity decreased by 26% in both surface to in vivo and control to in vivo defects. This model allows to study the regenerated bone without mechanically perturbing healing surface.