ABSTRACTAlizarin red S was extracted from aqueous media using an ionic liquid-based biphase system, including dipotassium hydrogen phosphate and 1-butyl-3-methyl-imidazolium chloride. The ionic liquid and kosmotropic salt concentrations were characterized for partitioning of the dye. The determination of alizarin red S was performed by high-performance liquid chromatography on a C18 column using 50% methanol and 20 mM phosphate buffer at pH 3 with external standard calibration from 20 to 100 mg/L. Spectrophotometric detection was performed at 255 nm. The limit of detection was 38.5 ng/mL. An extraction efficiency exceeding 95% was obtained. The key parameter to optimize the extraction efficiency and the preconcentration factor was the proportion of ionic liquid to the kosmotropic salt. An inverse relationship between the preconcentration factor and alizarin red S recovery was observed. The extraction efficiency increased with temperature by approximately 5% per 10°C.