The derivatization with NaBEt(4) for the determination of butyltin compounds in mussel tissues (Mytilus galloprovincialis) by GC-MS was optimized using a central composite design. The effects of NaBEt(4) concentration, pH and acetate buffer concentration on the derivatization efficiency were considered. Solid-phase extraction with Florisil cartridges was performed, demonstrating that the clean-up drastically reduces the background and improves the sensitivity. The good accuracy of the method was verified on a certified reference material (ERM 477); the figures of merit for all the three analytes, evaluated under optimum conditions, were satisfactory. The optimized derivatization procedure was applied to the determination of the analytes in mussels exposed to tributyltin (TBT). All considered tissues showed considerable accumulation of TBT, especially gills.