Background: Drug utilization evaluation (DUE) was performed to assess using drugs with special conditions such as antibiotics. Vancomycin is one of the essential antibiotics that is effective on methicillin-resistant Staphylococcus aureus, but unreasonable use of vancomycin, in addition to cost, leads to outbreak microbial resistance, which is a concern for health care systems all around the world. The aim of this study was to evaluate vancomycin use in teaching hospitals in Hamedan, Iran. Methods: This retrospective study was performed on patients who received vancomycin for at least 72 hours in intensive care units and infectious wards between April and September 2020 in the teaching hospitals of Hamedan. Data were obtained from patients’ medical records and entered into predesigned checklists. Results: From 661 patients, 441 were males and 247 were females. They received vancomycin for an average of 5.75 days. The most prevalent indication for vancomycin use was surgery. Only 40 patients had sensitive microbial culture to vancomycin, while 356 of them (53.9%) had no microbial culture at all. According to HICPAC guidelines, the overall appropriate use of vancomycin was 44.8%. Conclusion: According to the results, the irrational use of vancomycin must be corrected to achieve maximum optimal use; thus, performing and adhering to microbial culture, deploying clinical pharmacists, and holding explanatory sessions for health care are recommended for this purpose.