In this work, an automated liquid-liquid microextraction procedure for the determination of sulfonamides (sulfamethoxazole, sulfamethazine and sulfapyridine) in urine samples using natural deep eutectic solvent is presented for the first time. The mechanism for extraction of sulfonamides was based on the formation of colored Schiff bases in the presence of vanillin, which acted as a derivatization reagent and precursor of natural deep eutectic solvent (an extractant). In this procedure, thymol was used as both media for Schiff bases formation and as a second precursor of the natural deep eutectic solvent. The formation of the Schiff bases was confirmed by mass spectrometry. A Lab-In-Syringe concept was applied for the automation of the microextraction procedure. The procedure involved mixing the sample and natural deep eutectic solvent into a syringe of a flow system, formation and microextraction of colored Schiff base followed by UV–Vis detection. Under optimal automated conditions the limits of detection, calculated from a blank test based on 3s (sigma) were 0.06, 0.1, and 0.06 mg L⁻¹ for sulfapyridine, sulfamethoxazole and sulfamethazine. The proposed automated procedure permitted the routine determination of one drug (sulfamethoxazole, sulfamethazine or sulfapyridine) in urine samples to be achieved in less than 10 min.