Nanoparticles have gained popularity in recent days due to their varied applications. Precise characterization of silk fibroin nanoparticles (SFNs) is critical for advanced biomedical applications. A clear understanding of the different characteristics of silk nanoparticles helps in smooth translation towards advanced materials and biomedical devices. This chapter discusses about various analytical techniques employed for characterizing the high-demand and dynamic silk nanoparticles focusing on their size, morphology, secondary structure, surface charge, and physical properties. Key methods include DLS, SEM, TEM, AFM for size and morphology analysis, FTIR, XRD, CD spectroscopy, Raman spectroscopy, NMR for secondary structure analysis; contact angle measurement, X-ray photoelectron spectroscopy for determination of surface charge; TGA, DSC for thermal properties and UV–Vis spectroscopy, fluorescence spectroscopy for optical properties. In the majority of cases, the physical attributes of SFNs are often assessed employing two or more methods. The strengths and limitations of each technique make it difficult to choose the best method, often leading to a combinatorial method of characterization. Overall, this chapter focuses on providing a problem-solving approach by emphasizing the effective methods required for characterization of SFNs and tailoring their properties for targeted applications.