We have developed a 3-phase method for dispersive liquid-liquid microextraction of ß-lactam antibiotics in milk. Chloroform and acetonitrile serve as the solvents for extraction and disperssion, respectively, where Aliquat 336 is the carrier. An experimental design based on Plackett-Burman and Central composite designs were applied for the screening and optimization of significant parameters in the extraction method. The experimental conditions for extraction were optimized, and the subsequent HPLC assay gave relative standard deviations and detection limits in the range of 4.3–8.5 % and 50–500 μg L-1, respectively. Preconcentration factors are in the range of 80–125.