Mivacurium is a muscle relaxant that is used in hospitals and has been implicated in a number of homicides. A validated, sensitive method for the analysis of mivacurium and metabolites from biological specimens is presented here. Sample cleanup involves the precipitation of proteins with acetonitrile followed by analyte isolation using a commercially available solid-phase extraction column. Screening is performed by liquid chromatography with fluorescence detection, and confirmation is performed by liquid chromatography with tandem mass spectrometric detection. This method has been used to analyze dozens of forensic samples, including clinical specimens from living patients, as well as autopsy specimens from exhumed bodies.