Rapamycin is a multi-functional medication with an endless list of clinical activities and impressive potency. Regarding its remarkable high price, efforts intending rapamycin production from low-cost natural substrates are highly appreciated in the framework of its economic production. Ten natural additives were tested one at a time in a basal medium of natural substrates to potentiate the overproduction of the drug. The results were analyzed by single-factor ANOVA (at a 95% confidence level) which proved a very significant variation in rapamycin concentration (p-value=8.592x10-8). A significant increase in rapamycin yield from a base of 11.2 mg/l in the control to 15.1 and 15.6 mg/l was recorded in the case of oat bran and semolina verifying more than 34% and 39% increase in yield, respectively. Various supplements of different precursors, stimulators, and carbon sources were also tested, and markedly higher outputs were obtained with D-(+)-mannose, fructose, glycerol, L-tyrosine, and L-lysine. To get the optimal formulation of all-natural and additional substrates, two-step statistical designs were employed. Significant models of Plackett-Burman and Box-Behnken (p-value 0.0017 and 0.0003, respectively) revealed a formula that potentiated more than a 70% increase in drug production.