Chemical reactions of reducing agents in the gold nanoparticle (AuNP) formation process were characterized using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). As the reaction of the AuNPs progresses, the produced AuNPs can serve as an efficient SALDI substrate. SALDI-MS revealed that the reducing agents and their oxidation products can be determined in the mass spectra. With respect to the transmission electron microscopic and UV-Vis spectroscopic examination of AuNPs, SALDI-MS results confirm not only the tendency toward AuNPs formation, but also reflect the information of the redox reaction process. Our results provide useful information for developing SALDI-MS methods to explore the chemical information regarding the surface behavior between adsorbates and nanomaterials.