We have developed and validated a method for the quantification of fentanyl, a synthetic opioid, in dog plasma by on-line SPE with a hydrophilic column coupled to tandem mass spectrometry in positive electrospray mode. A column-switching instrument with 10-port valve and two HPLC pumping systems were employed. Deuterated fentanyl served as the internal standard. A Waters Oasis HLB extraction column and a Waters Atlantis HILIC Silica analytical column in a column-switching set-up with gradient elution were utilized. Both fentanyl (analyte) and the internal standard (fentanyl-d5) were determined via multiple reaction monitoring (MRM) and the MS/MS ion transitions monitored were m/z 337.0/188.0 and 342.0/188.0, respectively. Each plasma sample was chromatographed within 5 min. The calibration curves were linear over a widely range of 0.01-50 ng/mL using weighted linear regression analysis (1/x). The low limit of quantitation was 0.01 ng/mL. The intra- and inter-day accuracy ranged from 102 to 112% and the overall precision was less than 3%. The recoveries ranged from 90 to 105% in plasma at the concentrations of 0.04, 0.4, 4 and 40 ng/mL. No influence of freeze/thaw and long-term stability were observed. This validated method has been successfully applied to analyze the dog plasma samples of a pharmacokinetics study.