Optical spectroscopy methods have had considerable impact in the field of biomedical diagnostics, providing novel methods for the early or noninvasive diagnosis of various medical conditions. Among them, fluorescence spectroscopy has been the most widely explored mainly because fluorescence is highly sensitive to the biochemical makeup of tissues. It has been shown that tumors were easily detected on account of altered fluorescence properties with respect to fluorescence of ordinary tissue. Breast cancer is one of the most commonly diagnosed cancers among women in the world and also it is one of the leading causes of deaths from cancer for the female population. However, when detected in early stage, it is one of the most treatable forms of cancer. Therefore, fluorescence technologies could be highly beneficial in early detection and timely treatment of cancer. This chapter presents main results and conclusions that have been reported on the use of fluorescence spectroscopy for the investigation of breast cancer. It also gives an overview on the instruments and methodology of measurements, on the main endogenous fluorophores present in tissues, on the tissue fluorescence, and on the statistical methods that aid interpretations of fluorescence spectra. Finally, examples of using various fluorescence techniques, such as excitation, emission and synchronous spectroscopy, excitation-emission matrices, and lifetimes, for the breast cancer diagnosis are presented.