Bone healing involves compositional changes and understanding these is potentially important for prevention and treatment of bone diseases like osteoporosis. The health and proper function of bone tissues depends crucially major elements like calcium and phosphorus, but also on trace elements like iron, zinc, and strontium. This work employs energy dispersive X-ray fluorescence spectroscopy (μ-EDXRF) for trace element analysis, particularly of iron, and Raman spectroscopy for analysis of collagen, during healing of subcritical calvarial defects. In vivo defects were created on the calvaria of Sprague-Dawley rats (n=16) using a 1.4 mm burr drill. Subjects were sacrificed and additional control defects were similarly created after 7 (n=8) and 14 days (n=8) of healing. The two spectroscopy methods analyzed the bone surface at both time points and defect types without mechanical perturbation. Compared to control defects, calcium was found significantly decreased while zinc and iron increased in in vivo defects. Iron increased 6.7fold after 7 days, and this increased reduced approximately 50% after 14 days. Raman showed decreased collagen alignment after 7 days, which became insignificant after 14 days. We deduce that new collagen formation during healing, as revealed by Raman spectroscopy, scanning electron and optical microscopy and surface profiling, resulted in trace element changes. Our results show the need for studying the concentrations of major and trace elements, with iron in particular playing a crucial role in healing.