Norvancomycin (NVCM) is used in treating patients with infections caused by drug-resistant gram-positive bacteria. The NVCM plasma concentration varies greatly among individuals. To avoid the adverse drug reactions and prevent the presence of drug-resistant bacteria, the routine monitoring of NVCM in blood plasma was strongly recommended. However, there were few methods for plasma NVCM analysis. To the best of our knowledge, this is the first validated ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for plasma NVCM analysis. The ion transition for NVCM was m/z 718.5 > 144.1. The flow rate was 0.4 mL/min with a run time of 3.5 min. The calibration range of the UPLC-MS/MS method was 1-100 mg/L. The intra-day and inter-day inaccuracy and imprecision were -7.39%-10.27% and less than 11.55%. The internal standard (IS) normalized recovery and matrix factor were 68.24%-78.24% and 94.53% to 115.80%, respectively. The coefficient variations of IS normalized recovery and matrix factor were less than 11.24% and 9.14%. Age, body weight, and creatinine clearance affect NVCM plasma concentrations in 20 patients. Further studies are warranted to confirm the results.