Background & Objectives: Vancomycin, as β-lactam classes of antibiotics, is widely used for treating infectious diseases in hospitals. Therefore, it is necessary to establish a method for precise measurement of vancomycin in hospital sewage as an emerging pollutant that causes bacterial resistance. Methods: In this study, solid phase extraction was used for separation of vancomycin from hospital wastewater. Vancomycin recovery rate were investigated at pH 3 and 7. The extracted samples were passed through a C18 column at 20 °C using different ratios of mobile phase of acetate/acetonitrile buffer with flow rate of 0.8 ml min-1. The quantity of vancomycin in samples was detected by high performance liquid chromatography (HPLC) equipped with UV detector at 240 nm wave length. Results: The best condition for extraction was achieved at pH=7 with a recovery of 89%. Better results were obtained by mobile phase of acetate/acetonitrile buffer with volume ratio of 89:11 at pH=4. The linearity coefficients (R2) of 0.97 and 0.96 were obtained for vancomycin concentration ranges of 1-45 and 50-120 µg l-1 at optimum condition, respectively. Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated as 3 and 10 µg l-1 respectively. Also, variations in precision of both hourly and daily measurements of standard solution were less than 10%. Conclusion: The results of this study might be applied as a specific method for extraction and analysis of vancomycin antibiotic in hospital sewage samples.