A simple and efficient new procedure is presented for the analysis of diflubenzuron (DFB) accumulation in tilapia (Oreochromis niloticus) filet. A liquid-chromatography (LC) with diode array detection method with C(18) solid-phase extraction clean-up was employed. The methods exhibit no significant matrix effect as verified by the recovery efficiency. The limits of detection and quantification were 32 microg/kg and 110 microg/kg, respectively. LC-tandem mass spectrometry analysis confirmed the presence of DFB in filet of tilapia exposed to this pesticide. The method was successfully applied for the analyses of fish captured in three different fee-fishing farms during two seasons and for the analyses of fish from an experimental pond (subjected to Dimilin exposition) and depuration tank during different time intervals.