This work proposes a new methodology to identify the best medium concentrations for fed-batch production of hairy root using Datura innoxia as a model. Firstly, the role of each component on the growth rate is investigated separately. Then, an experimental design allows refining the optimization studying the interactions between the major species. The result analysis let to define concentration range optimized for fed-batch process. The work novelties lie in two aspects. Firstly, concentrations have been kept constant during each run. Thus, biomass uptakes do not affect the optimization and the growth rate is maintained constant during the exponential phase. Secondly, the effects of salts are generally studied. In this work, the influences of each ion are investigated in order to avoid bias due to the counter-ion effects. Compared to the classical B(5) medium, the optimized medium shows a significant improvement leading to more than 80% increase of final biomass production.