Raman and Fourier transform infrared (FT-IR) spectroscopic imaging techniques can be used to characterize bone composition. In this study, our objective was to validate the Raman mineral:matrix ratios (ν1 PO4:amide III, ν1 PO4:amide I, ν PO4:Proline + hydroxyproline, ν1 PO4:Phenylalanine, ν1 PO4:δ CH2 peak area ratios) by correlating them to ash fraction and the IR mineral:matrix ratio (ν3 PO4:amide I peak area ratio) in chemical standards and native bone tissue. Chemical standards consisting of varying ratios of synthetic hydroxyapatite (HA) and collagen, as well as bone tissue from humans, sheep, and mice, were characterized with confocal Raman spectroscopy and FT-IR spectroscopy and gravimetric analysis. Raman and IR mineral:matrix ratio values from chemical standards increased reciprocally with ash fraction (Raman ν1 PO4/
Amide III: P < 0.01, R2 = 0.966; Raman ν1 PO4/Amide I: P < 0.01, R2 = 0.919; Raman ν1 PO4/Proline + Hydroxyproline: P < 0.01, R2 = 0.976; Raman ν1 PO4/Phenylalanine: P < 0.01, R2 = 0.911; Raman ν1 PO4/δ CH2: P < 0.01, R2 = 0.894; IR P < 0.01, R2 = 0.91). Fourier transform infrared mineral:matrix ratio values from native bone tissue were also similar to theoretical mineral:matrix ratio values for a given ash fraction. Raman and IR mineral:matrix ratio values were strongly correlated (P < 0.01, R2 = 0.82). These results were confirmed by calculating the mineral:matrix ratio for theoretical IR spectra, developed by applying the Beer–Lambert law to calculate the relative extinction coefficients of HA and collagen over the same range of wavenumbers (800–1800 cm–1). The results confirm that the Raman mineral:matrix bone composition parameter correlates strongly to ash fraction and to its IR counterpart. Finally, the mineral:matrix ratio values of the native bone tissue are similar to those of both chemical standards and theoretical values, confirming the biological relevance of the chemical standards and the characterization techniques.