PURPOSE: The purpose of this study was to develop a protocol to prepare buffered chlorhexidine (CHX) eye drops (0.2% w/v) in the United Kingdom that can be reproduced at a production facility in Uganda. Buffered CHX eye drops can prevent CHX degradation and improve ocular tolerability during the treatment of fungal keratitis. METHODS: Buffered CHX eye drops in amber glass containers were prepared using sodium acetate buffer at pH 5.90 to 6.75. Two commercial CHX solutions and CHX in water were used as controls. Eye drops were stored at 40°C (70% humidity, 21 months) in the United Kingdom and at ambient temperature in Uganda (30 months). High-performance liquid chromatography was used to determine CHX stability over time, and pH was monitored. Sterility was achieved using an autoclave (121°C, 15 minutes) and water bath (100°C, 30 minutes). RESULTS: The pH of acetate-buffered CHX eye drops did not change over 21 months at 40°C or at ambient temperature (30 months), whereas the pH of the unbuffered aqueous CHX displayed significant fluctuations, with an increase in acidity. The CHX concentration remained the same in both buffered and unbuffered eye-drop solutions. Eye drops sterilization was successful using an autoclave and a water bath. CONCLUSIONS: Stable, sterile, buffered CHX eye drops (pH 6.75) were successfully prepared first in the United Kingdom and then reproducibly in Uganda. This eye drops can be prepared in a hospital or pharmacy setting with limited resources, thus providing a cost-effective treatment for fungal keratitis. TRANSLATIONAL RELEVANCE: A protocol has been developed to prepare buffered CHX eye drops in low- and middle-income countries to treat fungal keratitis.