Vancomycin is a critical antibiotic frequently utilized in clinical settings, with therapeutic drug monitoring (TDM) strongly advised to optimize treatment efficacy and mitigate the risk of adverse effects. However, current methods for measuring vancomycin levels in human plasma are hindered by long analysis times and complicated sample preparations. Thus, this study developed and validated a novel UPLC-MS/MS method for a rapid (with a running time of 3.5 min) and simple analysis of plasma vancomycin. To quantify vancomycin concentration in human plasma, we have developed and validated the UPLC-MS/MS method with high sensitivity, specificity, and accuracy, meeting the strict criteria according to the Food and Drugs Administration (FDA) guidelines for validation biological analysis methods. Vancomycin and atenolol (internal standard) underwent positive electrospray ionization (ESI+) and detection in multi-reaction monitoring (MRM) mode. The selected MRM transitions were m/z 725.66→144.16 for vancomycin and m/z 267.29→189.96 for atenolol. Plasma samples were precipitated using a simple mixture containing acetonitrile, methanol, and formic acid as a pH adjuster. The separation was performed using the Poroshell 120 Phenyl Hexyl Column (4.6 × 150 mm, 2.7 μm) maintained at 25 °C for 3.5 min. Isocratic elution with a mobile phase (methanol and 0.1% formic acid in a 40:60 v/v ratio) at a flow rate of 0.5 mL/min was employed. The method showed linearity (0.1–75 μg/mL) with a coefficient of determination above 0.9994 and a lower limit of quantification at 0.1 μg/mL. Precision, both intraday and interday, was below 10%, and accuracy ranged from 91.70% to 111.57%. System suitability, selectivity, stability, carryover, dilution, recovery, and matrix effect validation results all met acceptable criteria. The established UPLC-MS/MS method is expected to be a rapid, simple, and reliable tool for drug monitoring and pharmacokinetic studies, enhancing patient care during vancomycin administration.