Natamycin and Voriconazole are commonly used antifungal agents to treat fungal keratitis. Recently, the combination of these drugs has been proven synergistic in treating fungal keratitis. However, no simultaneous estimation method has been reported for Natamycin and Voriconazole using reverse-phase high-performance liquid chromatography (RP-HPLC). Hence, this study aims to develop an accurate, precise, reproducible, sensitive, and rapid HPLC method. The developed HPLC method was validated according to United States Food and Drug Administration (USFDA) guidelines. Chromatographic separation was performed using a Zorbax RX-C8 5 μm column with a dimension of 4.6 × 150 mm