Response surface methodology was used for modeling and optimizing microwave-assisted extraction of antioxidants from Thymbra spicata L. as a factor of temperature, extraction time, solvent concentration, and solvent-to-solid ratio. The prepared extracts showed maximum antioxidant properties, including total phenolic content (TPC), total antioxidant capacity (TAC), and radical scavenging activity (RSA) at the optimum operating conditions. All models calculated for the three responses that are TPC, TAC, and RSA were noteworthy (p < 0.0001) and showed a significant relationship between the response and independent parameters. There was a close relationship between the experimental and the predicted values obtained using the proposed method. The phenolic antioxidant profile of Thymbra spicata L. extract was characterized with the UPLC-PDA-ESI-MS/MS system and rosmarinic acid was found as a major component (1089.2 +/- 10.9 mg/100 g-DS). In the future, this optimized and modeled MAE method can be applied in food and pharmaceutical industries to effectively extract antioxidants from edible Thymbra spicata L. plant.