A novel approach for liquid-liquid microextraction via formation of hydrophobic deep eutectic solvent by pH adjusting was suggested. This study demonstrated for the first time that an alkaline aqueous solution of terpenoid and medium-chain saturated fatty acid can serve as a source of precursors for deep eutectic solvent (DES) formation by extraction system acidification. In this case, the microdroplets of in situ formed DES phase appear in the whole volume of the homogeneous solution, simultaneously extracting the analytes. The proposed approach enables one to avoid heating and usage of auxiliary energy sources, which considerably simplifies the procedure and minimizes the extraction time in comparison with the existing analogues. The proposed approach was successfully applied to the determination of sulfamethazine, sulfamethoxazole, sulfaquinoxaline and sulfadiazine in milk samples. The nature of precursors played an important role in the extraction process, providing different types of interaction with the analytes. The preconcentration factors ranged from 22 to 103, rising with an increase of hydrophobicity of the analyte. In situ formation of fine droplets of the solvent provided fast mass-transfer of sulfonamides (30 s). The limits of detection calculated from the calibration plot based on 3σ were equal to 1 μg L⁻¹ for sulfamethazine and sulfaquinoxaline, 3 μg L⁻¹ for sulfamethoxazole, and 5 μg L⁻¹ for sulfadiazine.