Vancomycin is an antimicrobial agent that exhibits high efficacy against Gram-positive bacteria. The importance of therapeutic drug monitoring (TDM) for vancomycin has been substantiated in specific patient cohorts, underscoring the significance of determining vancomycin plasma levels. This study presents the development and validation of a simple, reproducible, and practical approach for quantifying vancomycin levels in human plasma samples through high-performance liquid chromatography (HPLC). Deproteinization of plasma samples (0.3 mL) was achieved using 10% perchloric acid. The chromatographic separation was achieved using a C18 column. The mobile phase, consisting of phosphate buffer and acetonitrile (90:10, v/v), was run at a flow rate of 1 mL/min. Ultraviolet detection was conducted at a wavelength of 192 nm and the method was linear in the range of 4.5–80 mg/L (r² > 0.99). Inter- and intra-day assay precision and accuracy were determined to be within the acceptable range. The run time was noted to be 10 min. This method was evaluated using different greenness tools, which indicated that the method is environmentally friendly. Our method was effectively applied to analyze vancomycin concentrations in critically ill patients. Thus, our approach has the potential for practical implementation in routine TDM procedures.