A simultaneous determination of sulfamonomethoxine, sulfadimethoxine, and their hydroxy/N(4)-acetyl metabolites in chicken plasma, muscle, liver, and eggs using gradient high-performance liquid chromatography (HPLC) with a photo-diode array detector is developed. All the compounds are extracted by a handheld ultrasonic homogenizer with ethanol followed by centrifugation. The separation is performed by a reversed-phase C4 column with a gradient elution (ethanol:1% (v/v) acetic acid, v/v; 10:90-->20:80). Average recoveries from samples spiked at 0.1-1.0mugg(-1) or mugml(-1) for each drug were >90% with relative standard deviations within 4%. The limits of quantitation were <30ngg(-1) or ngml(-1).