The ability to probe fresh tissue is a key feature to biomedical Raman spectroscopy. However, it is unclear how Raman spectra of calcified tissues are affected by freezing. In this study, six transverse sections of femoral cortical bone were subjected to multiple freeze∕thaw cycles and probed using a custom Raman microscope. Significant decreases were observed in the amide I and amide III bands starting after two freeze thaw cycles. Raman band intensities arising from proline residues of frozen tissue appeared consistent with fresh tissue after four cycles. Crystallinity values of bone mineral diminished slightly with freezing and were noticeable after only one freezing. Mineral carbonate levels did not deviate significantly with freezing and thawing. The authors recommend freezing and thawing bone tissue only once to maintain accurate results.