An isocratic reversed-phase ion-pair liquid chromatography with UV detection at 350 nm for the determination in human plasma of floctafenin (F) and its three main metabolites—floctafenic acid (FA), hydroxyfloctafenin (HOF), and hydroxyfloctafenic acid (HOFA)—is reported. Analytes and internal standard were extracted from acid plasma into ethyl acetate, and this organic phase was evaporated to dryness. This extraction yielded plasma drug recoveries of >72%. Using 1 ml of plasma, the lower quantification limit was 0.05 mUg ml-1with excellent linearity up to 0.8 mUg ml-1for HOF and HOFA and up to 4.0 mUg ml-1for F and FA. The reproducibility and the selectivity of the method for several drugs thought likely to be administered in conjunction with F, were demonstrated. This method has been successfully applied to a pharmacokinetic study with a single 10 mg kg-1oral dose in ten children.