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Magnetite nanocrystal clusters are being investigated for their potential applications in catalysis, magnetic separation, and drug delivery. Controlling their size and size distribution is of paramount importance and often requires tedious trial-and-error experimentation to determine the optimal conditions necessary to synthesize clusters with the desired properties. In this work, magnetite nanocrystal clusters were prepared via a one-pot solvothermal reaction, starting from an available protocol. In order to optimize the experimental factors controlling their synthesis, response surface methodology (RSM) was used. The size of nanocrystal clusters can be varied by changing the amount of stabilizer (tribasic sodium citrate) and the solvent ratio (diethylene glycol/ethylene glycol). Tuning the experimental conditions during the optimization process is often limited to changing one factor at a time, while the experimental design allows for variation of the factors’ levels simultaneously. The efficiency of the design to achieve maximum refinement for the independent variables (stabilizer amount, diethylene glycol/ethylene glycol (DEG/EG) ratio) towards the best conditions for spherical magnetite nanocrystal clusters with desirable size (measured by scanning electron microscopy and dynamic light scattering) and narrow size distribution as responses were proven and tested. The optimization procedure based on the RSM was then used in reverse mode to determine the factors from the knowledge of the response to predict the optimal synthesis conditions required to obtain a good size and size distribution. The RSM model was validated using a plethora of statistical methods. The design can facilitate the optimization procedure by overcoming the trial-and-error process with a systematic model-guided approach. Full article