Vancomycin (VCN) is a glycopeptide antibiotic widely used to treat Gram-positive infections such as those caused by methicillin-resistant Staphylococcus aureus (MRSA). Its narrow therapeutic window, high interindividual pharmacokinetic variability, and dose-dependent nephrotoxicity support the need for therapeutic drug monitoring (TDM). In this study, a UHPLC-UV method for VCN quantification in human plasma was developed and validated according to ICH M10 guidelines, using a one-step protein precipitation requiring only 100 µL of plasma. Clinical applicability was preliminarily explored in three hospitalized patients, where the area under the concentration-time curve over 24 h to minimum inhibitory concentration ratio (AUC₂₄/MIC) was estimated using PrecisePK, NextDose, and TDMx Bayesian software platforms before and after dose adjustment. The method showed linearity from 0.5 to 100 µg/mL (r² > 0.99), with accuracy within ±7% and precision below 11%. Acceptance criteria for selectivity and matrix effect were met, and stability was confirmed under multiple conditions, including clinically relevant storage and transport. Pre-adjustment AUC₂₄/MIC values were outside the target window of 400–600 in all patients, confirming the pertinence of implementing model-informed precision dosing as a routine TDM practice in Colombian health institutions.