A simple method was developed for determining florfenicol concentration in a small volume (250 μl) of plasma from five phylogenetically diverse species of freshwater fish. Florfenicol was isolated from the plasma matrix through C18 solid-phase extraction and quantified by reversed-phase high-performance liquid chromatography with UV detection. The accuracy (84–104%), precision (%RSD⩽8), and sensitivity (quantitation limit <30 ng/ml) of the method indicate its usefulness for conducting pharmacokinetic studies on a variety of freshwater fish.