Hydrophilic 3-hydroxypyridin-4-ones (HPOs), such as 1-(2'-carboxyethyl)-2-methyl-3-hydroxypyridin-4-one (CP38), 1-(3'-hydroxypropyl)-2-methyl-3-hydroxypyridin-4-one (CP41) and 1-(2'-hydroxyethyl)-2-ethyl-3-hydroxypyridin-4-one (CP102), are orally active iron chelators and ester prodrugs of these molecules are currently under investigation. A liquid extraction method using acetonitrile and 2-propanol (80:20 v/v) under acidic and NaCl-saturated conditions has been developed in order to efficiently extract these HPOs from various matrices. The extracted HPOs were determined using a reversed phase polymer HPLC column (PLRP-S 100 A) and the gradient ion-pair mobile phase containing tetrabutylammonium chloride (5 mM) and EDTA (0.5 mM). The extraction recovery of these chelators in phosphate buffer, rat blood and liver homogenate varied from 85 to 94%. The coefficients of variation (C.V.) for within-day determination were in the range of 1.4-3.3% at 1 mM and 2.0-4.7% at 0.1 mM. High accuracy of determination was also achieved.