Nanotechnology has led to the development of new and improved materials, and particular emphasis has been directed toward nanoparticles and their multiple bio-applications. Nanoparticles exhibit size-, shape-, and composition-dependent properties, e.g., surface plasmon resonance and photothermal properties, which may potentially enhance laser desorption/ionization systems for mass spectrometry-based analysis of biomolecules. Also, nanoparticles possess high surface to volume ratio that can be easily derivatized with a wide range of ligands with different functional groups. Surface modification makes nanoparticles advantageous for sample preparation procedures prior to detection by mass spectrometry. Moreover, it allows the synthesis of affinity probes, which promotes interactions between nanoparticles and analytes, greatly enhancing the ionization efficiency.This chapter provides a comprehensive discussion on the use of nanoparticles for mass spectrometryrelated applications, from sample preparation methodologies to ionization surfaces. Applications will focus on nanoparticle size, composition, and functionalization, as a comparative point of view on optimal characteristics toward maximization of bioassay efficiency.