Determination of the stoichiometry of reactions is a pivotal step for any chemical reactions toward a desirable product, which has been successfully achieved in organic synthesis. Here, we present the first precise determination of the stoichiometry for the reactions toward gold nanoparticle formation in the sodium borohydride reduction method. Leveraging on the real-time mass spectrometry technique, we have determined a precise balanced reaction, 32/x [Au(SR)](x) + 8 e(-) = [Au-25(SR)(18)](+) 7 [Au(SR)(2)](-) (here SR denotes a thiolate ligand), toward a stoichiometric synthesis of water-soluble [Au-25(SR)(18)](-), where 8 electrons (from reducing agents) are sufficient to react with every 32 Au atoms, leading to the formation of high-purity [Au-25(SR)(18)](-). More interestingly, by real-time monitoring of the growth process of thiolate-protected Au nanoclusters, we have successfully identified an important yet missing byproduct, [Au(SR)(2)](-). This study not only provides a new method for Au nanocluster synthesis using only a stoichiometric amount of reducing agent in aqueous solutions (although the synthesis of organic-soluble Au nanoclusters might require a more delicate design of synthetic chemistry) but also promotes the mechanistic understandings of the Au nanocluster growth process.