Chitosan-based nanomaterials have shown rapid pace to occupy indispensable role in biological sciences due to their unique properties and functionalities distinct from bulk chitosan. This new family of nanomaterials is being developed for various applications like smart delivery of bioactive compounds, enhancing plant growth, boosting immune response, and controlling plant diseases. With an exponential growth in interest on these nanomaterials, development of measurement protocols for reliable, accurate, and consistent characterization is very important. With the advancement of characterization techniques, the physicochemical properties of chitosan-based nanomaterials such as size, shape, interactions among constituent components, surface charge, elemental composition, purity, crystallinity, surface morphology, and internal structure can be accurately determined. Further, newer analytical techniques allow the analysis of nanomaterials in their chemically unmodified and fully hydrated states. Characterization of nanomaterials provides progression in understanding, method optimization, and their applications with sustainability and accuracy. In this chapter, we have described some of the important characterization techniques for chitosan-based nanomaterials.