A fully automated in-syringe switchable hydrophilicity solvent-based microextraction approach was suggested for the first time. Di-(2-ethylhexyl)phosphoric acid was investigated as a novel switchable hydrophilicity solvent. The microextraction procedure implemented into a syringe pump included dissociation of the extractant in alkaline sample solution resulting in homogeneous solution formation followed by in situ organic phase generation by acidification and its separation. The microextraction procedure was applied to the HPLC-UV determination of antimicrobial drugs (sulfamethoxazole and sulfamethazine) in human urine samples as a proof-of-concept example. The calibration graphs were linear over the concentration ranges of 0.06–50 mg L−1 for sulfamethoxazole and 0.13–50 mg L−1 for sulfamethazine. The LODs calculated from the blank tests based on 3σ were 0.02 and 0.04 mg L−1 for sulfamethoxazole and sulfamethazine, respectively. The sample throughput was 12 samples h−1. The possibility of using the proposed procedure for assessing sulfamethoxazole/sulfamethazine acetylation in metabolic processes by individual human phenotypes was shown.