A simultaneous method was successfully established and validated for the separation and determination of buprenorphine (BP), its primary metabolite, nor-buprenorphine (NBP) and a proposed co-formulate, naloxone (NLX) in human plasma. The method used buprenorphine-d 4 (BP-D 4 ), nor-buprenorphine-d 3 (NBP-D 3 ), naltrexone (NTX) as internal standards (ISs). 100 µL of plasma sample fortified with the ISs was cleaned up by solid-phase extraction (SPE), and was then separated on a Waters Acquity TM BEH C 18 column with gradient elution using methanol and water (containing 0.2% formic) at a flow rate of 0.25 mL•min -1 . The mass spectrometer was used for detection and was operated in the positive electrospray ionization with multiple reaction monitoring (MRM) mode. The three compounds were effectively separated in 5 min. The linear ranges of the compounds were 0.1-25, 0.25-25 and 0.05-25 ng•mL -1 for BP, NBP and NLX, respectively, with r≥0.9935. The method had high sensitivity (the limits of detection were 0.02, 0.1 and 0.01 ng•mL -1 for BP, NBP and NLX, respectively) and high recoveries (≥97.6%). The result was shown to be linear and satisfactorily met current acceptance criteria for validation of bioanalytical method: intra and inter assay precisions within the required limits of ≤25% RSD. The LOQs fulfilled the LOQ requirements: precision≤25% RSD, and was fully validated according to the State Food and Drug Administration (SFDA) regulations. The results demonstrated that ultra-high performance liquid chromatographytandem mass spectrometer (UPLC-MS/MS) with SPE was a powerful detection tool and contributed to pharmaceutical analysis in biological matrices.