For the pretreatment of refractory sulfidic gold ores, processing gold-rich concentrates instead of ore offers several advantages, mainly enhancing cost-effectiveness of the process. In this study, an integrated approach combining experimental design and automated quantitative mineralogy (AQM) was developed to optimize gold flotation recovery from a refractory gold ore. Complete ore characterization using AQM and scanning electron microscopy (SEM) revealed that gold occurs as nanoscale inclusions (288.8–489.5 nm) within pyrite and arsenopyrite. Mineral mapping showed a composition dominated by silicates and minor sulfides. Optimal flotation conditions were determined using a Doehlert design, with pH and particle size distribution (D80) as variables. The optimized conditions (pH = 9.56, D80 = 88 µm) yielded a gold recovery of 86.76% and a concentrate gold grade of 18.80 ppm, compared to 2.61 ppm in the feed. Modal mineralogy and particle mapping indicated that the concentrate was enriched in sulfides (pyrite 58.9%, arsenopyrite 6.6%), while the tailing was mainly silicates (quartz 34.6%, albite 26.2%, muscovite 18%), confirming effective sulfide-gangue separation. Gold remaining in the tailing was enclosed in albite and quartz, demonstrating that the achieved recovery is ultimate, as this gold is non-floatable. The enhanced recovery and grade at higher pH were attributed to the improved stability of potassium amyl xanthate under alkaline conditions, as confirmed by Fourier Transform Infrared (FTIR) spectroscopy.