Lemon balm (Melissa officinalis) is a member of medicinal and aromatic plants that have been used for phytotherapeutic applications for decades. In this study, optimum production conditions of lemon balm tincture, which is a functional product used commonly by the people for medicinal purposes, were investigated. For this aim, the response surface methodology approach was applied, and to determine the effect of processing variables, solid concentration (X-1), ethanol concentration (X-2), and processing time (X-3) were selected as the parameters showing the essential effects on the tincture production. The production of high phenolic yielded tincture was aimed at, and therefore, total phenolic content (TPC) and antiradical activity as inhibition concentration (IC50) of the tinctures were determined as the responses in the study. According to the experimental design, 15 tincture samples were prepared, and TPC ranged 1,462-10,335 mg GAEL, while the IC50 ranged 11.8-78.1 mL. Multiple response optimization approaches were expertly applied, and optimum solid concentration and ethanol concentration for the high bioactive lemon balm tincture was determined as 5 gin 30 mL solvent and 38.6%, respectively. These optimum production conditions will help manufacturing the ideal lemon balm tincture with high bioactivity.