Bone composition, consisting of hydroxyapatite mineral and collagen matrix, can be altered by disrupted cellular remodeling processes and changes in serum chemistry, occurring with disease and drug treatments. Therefore, analysis of bone composition can contribute to the understanding of bone pathologies. FT-IR and Raman spectroscopy are two techniques to characterize the bone composition, although their outcomes are incompletely validated and understood. In addition, FT-IR and Raman spectroscopy can provide insight into the effects on bone tissue composition of active agents used to treat osteoporosis and diseases such as type 2 diabetes mellitus (T2DM), which are incompletely understood. Therefore, the objective of this dissertation was to validate FT-IR and Raman spectroscopic measures of bone composition and implement them to elucidate the role of osteoporosis treatments and T2DM on bone composition. In this dissertation, FT-IR and Raman bone mineral outcomes were validated against gold standard analytical measures in chemical standards and native tissue. Next, with a more complete understanding of these outcomes, Raman spectroscopy was applied in a pre-clinical murine study of anti-osteoporosis treatments. I demonstrated that sequential therapies decrease collagen maturity vs monotherapies, suggesting potentially improved bone material properties. Finally, FT-IR and Raman spectroscopy were applied in a translational study on clinical specimens from men with T2DM. Men with T2DM generally have similar bone composition, suggesting that the interaction between a robust cancellous compartment, characteristic to individuals with T2DM, and cortical bone may play a role in skeletal fragility in individuals with T2DM. The present work provides a quantitative foundation for researchers to apply FT-IR and Raman spectroscopy to characterize bone composition and demonstrates the ability of these techniques to contribute to the understanding of novel treatments for osteoporosis and skeletal fragility in men with T2DM.