During the past decades, several trials targeted a stable, sustainable and economic production of St. John’s wort (Hypericum perforatum) extract. The value of this extractstems from its use to treat depression and skin irritation due to its hyperforin con-tent. Previously, hyperforin-forming in vitro root cultures were established. Here, detailed growth and production kinetics have been analyzed over 40 days of culti-vation. In the first 10 days, sucrose was completely hydrolyzed to glucose and fruc-tose. The ammonium consumption supported the increase in the biomass and hyper-forin production. When sucrose was replaced with glucose/fructose, the linear growth phase started 6 days earlier and resulted in a higher space-time-yield. The maxi-mum hyperforin production was 0.82 mg L−1day−1, which was 67 % higher than in the sucrose-supplemented standard cultivation. Buffering the sucrose-supplemented medium with phosphate caused a 2.7-fold increase in the product to biomass yield coefficient. However, the combination of monosaccharides and buffering conditions did not cause an appreciable improvements in the production performance of the shake flask approaches. A potential scalability from flask to lab-scale stirred bioreactors has been demonstrated. The results obtained offer a basis for a scalable production of hyperforin and a sustainable source for a tissue culture-based phytomedicine.