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A simple liquid–liquid–liquid microextraction (LLLME) technique combined with liquid chromatography (LC) was developed for the extraction and quantitative determination of azithromycin (AZI) in biological fluids. In this technique, AZI was extracted from 2.5 mL basic sample solution (donor phase) at pH 10.5 into an organic phase (90 μL), for 30 min. Then back-extracted into a 5 μL microdrop acidic aqueous solution (acceptor phase) at pH 2.5 immersed in the organic phase from the tip of a microsyringe for 20 min with a stirring rate of 800 rpm and donor phase temperature of 35 °C. After a prescribed time, the acceptor microdrop was returned into the microsyringe and injected into the LC. Optimization of experimental parameters on LLLME efficiency was investigated. Under optimized conditions, a preconcentration factor of 85 and limit of detection of 0.03 μg mL⁻¹ were obtained. The calibration curve was …