A fentanyl patch is widely used for the treatment of cancer pain. Its few adverse effects include constipation and drowsiness. The absorption volume of transdermally applied fentanyl may differ according to its site of application and variability in patch adhesion. Since fentanyl is predominantly metabolized by the drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 in the liver, its concentration may vary in cases of physiologically reduced CYP3A4 activity in the liver (liver disease and aging) or on co-administration of drugs. The clinical significance of measuring plasma concentration of fentanyl is high, but conventional methods require complicated processes such as solid-phase extraction and liquid-liquid extraction before the sample is injected into an HPLC system. In this study, a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for determining plasma fentanyl concentrations by deproteinization with acetonitrile. A recovery test was conducted using an absolute calibration curve to confirm the method's linearity and inter- and intra-day reproducibility. The required plasma volume for detection was reduced from 1 mL in the conventional method to 20 µL in the present study, and a good calibration curve was obtained in the concentration range from 0.05 to 5 ng/mL. These findings suggest that the method for sample preparation and quantification developed in this study are appropriate for measuring fentanyl concentration in human plasma in clinical settings.