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The extract of the plant species Satureja hortensis L. (often used as traditional ethno-therapy and in food processing) was prepared using the ultrasonic extraction technique, and contains a large quantity of secondary metabolites, with scientific evidence for antioxidant, antimicrobial, sedative, antispastic and antidiarrheal activities. Process optimization was carried out using a mathematical–statistical method (response surface methodology—RSM), which models and examines the effects of three levels and three variables on the observed response. The investigated responses were the content of total phenolic components (TPC) and total flavonoids (TFC), as well as tests of antioxidant activity at the level of radicals. The independent variables were ethanol concentration (40–80%), temperature (40–80 °C) and the liquid–solid ratio (10–30 mL/g). Results from this study were entered into a second-degree polynomial with multiple non-linear regression. Analysis of variance (ANOVA) was applied to find the most favorable environment for assessing the model's performance and effectiveness with an ethanol concentration of 20%, temperature of 80 °C and LSR of 21.4 mL/g. ANOVA assessed the model's significance, and a second-order polynomial model described the relationships between variables and responses. Diagnostic plots confirmed the model's adequacy and reliability. The estimated values were 4.11 mg chlorogenic acid equivalents per gram of dry weight (CEA/g), 2.18 mg of rutin equivalents per gram of dry weight (RE/g), and 0.030 mg/mL and 0.030 mg/mL for TPC, TFC, IC50 and EC50, respectively. High-Performance Liquid Chromatography with diode array detection (HPLC-DAD) examination revealed that the prominent substance in the tested extract is rosmarinic acid (46,172 µg/mL), followed by chlorogenic acid (1519 µg/mL). [ABSTRACT FROM AUTHOR]