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Vancomycin is a glycopeptide antibiotic which contains a phenolic chromophore thus it absorbs radiations in the Ultra-violet range. This study was aimed to develop an analytical method for quantification of vancomycin in human serum. Vancomycin serial dilutions (200–12.5 µg/mL) were prepared in distilled water and studied for its UV absorption. Analytical method validation parameters like linearity, accuracy, precision, limit of detection and quantitation, and robustness were determined.% age recovery was studied from blank serum containing spiked drug levels after carrying out protein precipitation, ultracentrifuge drug extraction procedure and determining UV absorption. Post dose serum samples of 5 patients were studied for vancomycin levels. The method was found linear in the concentration range of 12.5 to 200 µg/mL. Correlation coefficient (R2) value was 0.99997 and the regression equation was (Y= 0.004 X+ 0.0055). The% RSD for accuracy and inter and intra-day precision was≤ 2.2%. The mean% age accuracy of 5 studied concentrations with five samples was 99.4%±1.6. The method was robust to changes in temperature from 4 to 40oC. The limit of detection and quantitation was found to be 0.99 and 3.02 µg/mL respectively. The mean% age recovery of spiked serum levels was 96.9%±2.17. The mean peak drug level in 5 patients was found to be 37.01±3 mg/L. Hence UV visible spectrophotometer can be used to study drug levels in serum samples.