The isolation and detection of pancuronium bromide was developed for aged autopsy samples to identify and confirm this compound in questioned tissue samples. A novel protocol was optimized for the isolation of the target drug in highly decomposed tissues. Solid-phase extraction (SPE) cartridges containing styrene-divinylbenzene were investigated. This polymer retained quaternary drugs and facilitated sequential elution upon washing with commonly available solvents. The semi-purified SPE samples were prescreened by pyrolysis GC-MS. A candidate specimen was then confirmed by microbore high-performance liquid chromatography/electrospray-ionization/mass spectrometry (microHPLC-ESI-MS/MS) with a triple-quadrupole mass spectrometer. The developed procedures provided a qualitative or semiquantitative (at best) basis for the investigation of difficult cases involving overdoses of polar drugs.