Background and purpose: Vancomycin is a glycopeptide antibiotic which is the drug of choice against methicillin-resistant Staphylococcus aureus. It has a narrow therapeutic index, and thus therapeutic drug monitoring (TDM), and clinical pharmacokinetic assessment are necessary in order to prevent adverse drug reactions such as nephrotoxicity. In this study, we aimed to develop a simple and validated HPLC method for vancomycin assay in order to establish a TDM center for patients admitted to the ICU of Nemazee Hospital in southern Iran. Experimental approach: In this study, a brief review of different parameters and variables which could affect the sensitivity, selectivity of the validated HPLC method for vancomycin determination were considered. According to the previous studies a simple, fast, and the relatively low-cost method was established for vancomycin determination in plasma samples. Findings/Results: The developed HPLC assay indicated a calibration curve with R-square of > 0. 999, acceptable selectivity, the accuracy of 90-105%, CV% of less than 15%, the limit of quantification of 1 µ g/mL, and limit of detection of 300 ng/mL. Vancomycin trough level, the area under the curve, renal clearance, the volume of distribution, , and elimination constant were measured in patients using this validated method. Conclusion and implications: Validated method for assay of vancomycin plasma levels was used to quantify vancomycin levels of four patients who were admitted to the ICU of Nemazee Hospital. According to the results, two of these patients showed lower levels than recommended therapeutic purposes while one of them showed a toxic level. According to the results, the TDM assessment of vancomycin is strongly recommended for patients who are hospitalized in ICU.