Purpose To evaluate the stability of 5% vancomycin ophthalmic solution prepared using balanced salt solution (BSS) and stored at –20°C in polypropylene containers. Methods Six batches of vancomycin 50 mg/mL eyedrops were aseptically prepared. One bottle of each batch was analyzed immediately after preparation, and the rest were stored at –20°C and analyzed using high-performance liquid chromatography (HPLC) at 30, 60, and 90 days to test their physicochemical stability and sterility. Thereafter, bottles were removed from the freezer and stored at 5°C for 30 days, with HPLC and other analyses repeated 105 and 120 days after preparation. All samples were analyzed in triplicate. Stability was defined as the absence of particles, color variation, or changes in pH and a remaining vancomycin concentration of 90% to 110% of the initial concentration. The sterility of the ophthalmic solution was evaluated by using soybean-casein digest broth with resins; samples were incubated for 7 days and checked daily for signs of microbial growth. Results There was no particle formation or sign of precipitation in any of the solutions throughout the duration of the study, regardless of the storage conditions. No change in color or turbidity was observed. The pH and osmolarity remained unchanged during storage at –20°C and after thawing. The vancomycin concentration remained within 10% of the initial concentration during the 90-day period of storage at –20°C and the subsequent 30 days after thawing. Sterility was preserved in all samples. Conclusion Vancomycin 5% prepared using BSS was physicochemically and microbiologically stable when stored at –20°C for 90 days. After thawing, this extemporaneous formulation remained stable when refrigerated at 5°C for 30 days.