Dynamic emergence of microbial keratitis (MK) requires a promising therapeutic arsenal of antifungal and antibacterial agents like voriconazole (VCZ) and moxifloxacin (MOXI), respectively. Parallelly, another paradigm of MK associated with ulcerative wounds cannot be left unnoticed and requires antifibrotic remedy (pirfenidone, PIR) as an authalic antimicrobial to retain the primordial vision. For designing an effective clinical cure, a combination of these three agents is required at a therapeutic dosage regimen. Following the quest, we have developed a simple and sensitive LC-MS/MS bioanalytical method for simultaneous quantification of VCZ, MOXI, and PIR in rabbit lacrimal fluid. The method was validated as per US-FDA norms using ketoconazole as an internal standard for linearity, accuracy–precision, matrix effect, dilution integrity, selectivity, and stability. The five minutes chromatographic set-up includes isocratic elution with a C18 column using MeOH (80%, v/v) and ultrapure water containing 0.2% formic acid (20%, v/v), respectively. The MS-based analyte detection was achieved in ESI⁺ multiple reaction monitoring mode. The sample extraction was performed using the protein precipitation method with minimal sample size. The validated methodology was employed to determine the ocular pharmacokinetics profile of marketed formulations containing VCZ, MOXI, and PIR in rabbit lacrimal matrix.