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,