<p>Hairy roots cultures resulting from transformation by the soil bacteria <i>Rhizobium rhizogenes</i> represent a biotechnological tool to produce specialized bioactive metabolites. We previously developed a protocol allowing the sustainable production of hairy roots from hybrid poplar 717-1B4 (<i>Populus tremula&#xa0;×&#xa0;Populus alba</i>). In the present study we assessed physicochemical parameters for the optimization of growth of hybrid poplar HP15-3 hairy root line and production of ethyl acetate extracts with antioxidant potential. The use of Woody Plant Medium (WPM) as mineral base significantly improved growth index by 2.1-fold compared to Murashige and Skoog (MS) medium and was selected even if a slight decrease in total phenolic content (TPC) and antioxidant activity could be observed (DPPH and FRAP assays). Among the different concentrations tested (15, 30 and 60 g·L⁻¹), sucrose at 30 g·L⁻¹ made it possible to significantly increase the growth index, without affecting the TPC and the antioxidant potential of the root extracts. Stressful conditions likely to affect the biosynthesis of specialized metabolites were also tested. The cultivation of hairy roots under LED light conditions resulted in a significant 2.45-fold increase in growth index. Antioxidant activity was also increased in relation to increased anthocyanin production. Furthermore, we demonstrated the feasibility of poplar hairy roots biomass production in temporary immersion bioreactors (RITA^®) under two water and mineral supply conditions. UHPLC/HRMS analysis of the extracts allowed to identify and quantify two phenolic glycosides (tremulacin and nigracin) whose concentrations were increased during cultivation under LED light. Two flavonoids (cyanidin and cyanidin-3-glucoside) were identified in light-grown root extracts. The parameters optimized during this research work could be successfully transposed to produce root biomass in bioreactors and under light to obtain bioactive ingredients for cosmetic formulations.</p>