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A three-phase hollow fiber microextraction technique based on carrier mediated transport combined with high performance liquid chromatography-ultra violet detection (HPLC-UV) was applied in the extraction and determination of dexamethasone sodium phosphate in biological samples. Dexamethasone sodium phosphate (DSP) was extracted from 7.5 mL of acidic solution (source phase) of pH 3 into an organic phase (n-octanol containing 5% (w v−1) of Aliquate-336 as carrier) impregnated in the pores of a hollow fiber and finally back extracted into 24 μL of a basic solution (pH = 9.5) located inside the lumen of the hollow fiber (receiving phase). The extraction took place due to the gradient of the counter ion from the source to the receiving phase. In order to achieve maximum extraction efficiency, different parameters affecting the extraction conditions were optimized. Under the optimized conditions, a …