This chapter involves the description and application of three advanced analytical techniques that are currently used to assess the potential of biomass for the production of biofuels, feeds, and chemicals. X-ray diffraction, FT-IR, and NIR may be used to study the structure of fibers in native biomass as well as changes during conditioning, pretreatment, and processing in a modern biorefinery. X-ray diffraction is used mainly to study the crystallinity of the samples based on the cellulose fraction which is one of the two major barriers for hydrolysis. FT-IR is used to get insight about the presence and interactions of main components of the fiber such as cellulose, hemicelluloses, and lignin. NIR is mainly used for a fast chemical characterization of the biomass and it is gaining a place to study changes caused by the pretreatments.