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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 separation was achieved using a C18 column with methanol and aqueous 0.2% formic acid (80:20, v/v) as a mobile phase with a total run time and flow rate of 4 min and 400 µL/ min, respectively. The FCZ and OFX were detected utilizing positive ion electrospray ionization (ESI) in multiple reactions monitoring mode. The tear sample extraction was carried out using simple deproteination using methanol. The systematic method validation was carried out according to USFDA regulatory guidelines for selectivity, linearity (r2>0.99), intra-day and inter-day precision and accuracy, matrix effect, dilution integrity, and stability. The validated bioanalytical method was successfully pertained to determine the pharmacokinetics profile of FCZ and OFX marketed formulation in preclinical rabbit tears.