Fluorescence and reflectance spectroscopy provide the ability to assess tissue structure and metabolism in vivo in real time, providing improved diagnosis of pre-cancerous lesions. Reflectance spectroscopy can probe changes in epithelial nuclei that are important in pre-cancer detection, such as mean nuclear diameter, nuclear size distribution and nuclear refractive index. Fluorescence spectroscopy can probe changes in epithelial cell metabolism, by assessing mitochondrial fluorophores, and epithelial-stromal interactions, by assessing the decrease in collagen crosslink fluorescence that occurs with pre-cancer. Thus, fluorescence and reflectance spectroscopy provide complementary information useful for pre-cancer diagnosis. Tissue engineering provides three-dimensional cell cultures that can be used to further explore the relationship between tissue structure and biological events important in cancer development and progression. In the future, improving our understanding of the biological changes that can be assessed using spectroscopy will not only improve optical techniques but also provide new tools to better understand cancer biology.