Fentanyl is a synthetic opioid used to treat intense chronic pain. In this study, the authors report detection and quantification of fentanyl in sweat and hair of a patient receiving fentanyl (25 μg/h) via a transdermal therapeutic system (TTS) for 22 days. Sweat was collected using sweat patches every night on days 13-21 of the therapy, and hair was collected 12 weeks after the end of the treatment. Detection and quantification was performed with liquid chromatography-tandem mass spectrometry using electron spray ionization in selected reaction monitoring mode. Alfentanyl was used as internal standard for quantification in hair and in sweat. Sodium ions have been used as endogenous internal reference for determination of volume of sweat excreted on each patch. Results show presence of fentanyl in both matrices. Fentanyl concentrations in sweat varied from 0.17 to 1.02 ng/μL, and time-resolved segmented hair analysis showed a maximum fentanyl concentration of 0.48 ng/mg of hair during the period of the therapy.