Gold nanoparticles (AuNPs) were synthesized in microfluidic mixers by means of response surface methodology (RSM). A reverse‐staggered herringbone micromixer was employed and reaction temperature, concentration ratio of reactants, Reynolds number, and pH of chloroauric acid were varied, with desired responses being particle size and peak intensity from UV spectroscopy. RSM was performed by simultaneously optimizing variable ranges to identify the best fit of polynomial equations to experimental data. Results revealed the individual and synergistic roles of each reaction variable on particle size and UV peak intensity, leading to identification of the largest design space. The effect of reaction variables on AuNP synthesis and particle size was confirmed in serpentine mixers.