Abstract: Tapentadol is a novel drug of opioid pain reliever, which is extensively metabolized primarily through conjugation. Tapentadol glucuronide and tapentadol sulfate are major drug-related metabolites in circulation. The objectives of this study were to develop a simple and rapid method to determine tapentadol and evaluate the effects of conjugated metabolites on tapentadol quantification using liquid chromatography with tandem mass spectrometry in dog plasma. The analyte and tramadol (IS) were extracted from plasma by protein precipitation with methanol, and chromatographied on a XDB C₁₈ (50 mm×4.6 mm, 1.8 μm) column using a mobile phase of methanol and 5 mmol·L^-1 ammonium acetate (0.01% ammonia). Mass spectrometric detection was performed using the m/z 222→121 transition for tapentadol and the m/z 264→58 transition for the internal standard tramadol, the m/z 398→m/z 121 transition for glucuronides conjugate and the m/z 302→m/z 222 transition for sulfate conjugate. Conjugated metabolites could undergo in-source conversion to generate an ion that interfered the quantification of tapentadol. Chromatographic separation was achieved to elimination interferences due to in-source conversion of the conjugated metabolites. The standard curves were demonstrated to be linear in the range of 0.100 to 20.0 ng·mL^-1 for tapentadol. The intra-and inter-day precisions were within 5.1%, and accuracy ranged from -3.2% to 0. This method was successfully applied to the pharmacokinetics of tapentadol hydrochloride sustained release tablets in Beagle dogs.