Most common viruses causing ocular infections are Herpes Simplex Viruses (HSV) type 1 and type 2, Cytomegalovirus (CMV), Varicella-zoster Virus (VZV), and few strains of Adenovirus. Diagnosis of these infections through clinical manifestations and using conventional methods has a number of limitations. The purpose of this study was to develop a multiplex Polymerase Chain Reaction (PCR) for simultaneous detection of all pathogenic viruses from ocular infections. Ten uniplex PCRs were standardized, two each for HSV type 1 (HSV-1) and type 2 (HSV-2), CMV, VZV, and Adenovirus. Various multiplexing combinations of above PCRs were put to finalize targets and reaction conditions enabling diagnosis of all in a single reaction. The uniplex and multiplex PCRs were run for known positive and negative controls, and samples from clinically suspected patients and healthy controls. Out of the 170 samples from suspected ocular infections, 24.7% were positive by uniplex PCR and 22.9% were correctly identified by multiplex PCR. None of the samples negative by uniplex PCRs was positive by the multiplex PCR. The sensitivity and specificity of multiplex PCR compared to the commonly used uniplex PCRs as gold standard was 92.86% and 100%, respectively. The prevalence of different viral pathogens was 13.5% for HSV-1, followed by 5.9% for Adenovirus, 2.4% for VZV, 1.8% for HSV-2, and 1.2% for CMV. The establishment of multiplex PCR has found immediate application in diagnosing ocular viral pathogens in a single reaction, thus saving time, manpower, and resources by fivefold.