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The expansion of polymicrobial keratomycosis (PMK) requires dynamic pharmacotherapy of antimycotics along with antibacterial agents such as fluconazole (FCZ) and ofloxacin (OFX). To effective clinical cure, different microbes require different dosage regimens. A responsive, selective, and fast method for estimation of FCZ and OFX in rabbit tears using high-performance liquid chromatography together with tandem mass spectrometry (LC-MS/MS) was established and validated using ketoconazole as an internal standard (IS). An isocratic separat