<jats:p>Neomycin production of <jats:italic>Streptomyces fradiae</jats:italic> NCIM 2418 was optimized by using response surface methodology (RSM), which is powerful mathematical approach comprehensively applied in the optimization of solid state fermentation processes. In the first step of optimization, with Placket-Burman design, ammonium chloride, sodium nitrate, L-histidine, and ammonium nitrate were established to be the crucial nutritional factors affecting neomycin production significantly. In the second step, a 2<jats:sup>4</jats:sup> full factorial central composite design and RSM were applied to determine the optimal concentration of significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the important nutrients for the maximum were obtained as follows: ammonium chloride 2.00%, sodium nitrate 1.50%, L-histidine 0.250%, and ammonium nitrate 0.250% with a predicted value of maximum neomycin production of 20,000 g kg<jats:sup>−1</jats:sup> dry coconut oil cake. Under the optimal condition, the practical neomycin production was 19,642 g kg<jats:sup>−1</jats:sup> dry coconut oil cake. The determination coefficient (<jats:italic>R</jats:italic><jats:sup>2</jats:sup>) was 0.9232, which ensures an acceptable admissibility of the model.