Nanomaterials have attracted much to the recent researcher’s interest as a rapidly growing class of materials for many applications. Especially biologically derived nanomaterials construct many application-oriented properties by simple way of fabrication. Characterization of such materials for their phase is considered to be an important aspect to define properties and applications of materials. Many techniques have been used to characterize the materials for their confirmation, physical properties, and nature of biocompatible nanomaterials. In several cases, more than one characterization technique are supposed to be used to evaluate the materials for their identification. It is a well-known fact that the fabrication of bioderived nanoparticles are constantly increasing, hence it is a challenging to the researchers for reliable characterization of nanomaterials for specialized applications. The aim of this chapter is to summarize the importance of experimental techniques for the characterization of biocompatible nanoparticles. Principles and instrumentation of some important characterization techniques are summarized. Also, the main characteristics of the techniques and their operation principles in characterization of biomaterials are described.