The short-acting synthetic opioid alfentanil undergoes extensive biotransformation to several metabolites. A gas chromatographic-mass spectrometric assay, using selected-ion monitoring and deuterated internal standards, was developed for quantitating the predominant metabolites of alfentanil. Optimal extraction and derivatization conditions are described. The assay was applied to the analysis of metabolites formed during alfentanil metabolism in vitro by human liver microsomes. Formation of known alfentanil metabolites was confirmed, and formation of a metabolite, not previously detected in vitro, is described. The assay represents a significant improvement over existing methods of alfentanil metabolite analysis, which use HPLC and radiochemical detection.