Infectious uveitis is a vision threatening inflammatory ocular disease wherein early diagnosis may prevent the loss of vision. The purpose of this study was to develop a multiplex real-time PCR for the diagnosis of Herpes simplex virus-1, Varicella zoster virus, cytomegalovirus and Toxoplasma gondii in patients with suspected infectious uveitis. A total of 126 intraocular samples (aqueous and vitreous humor) were collected and subjected to multiplex real-time PCR. Overall 26.2% (33/126) patients were found to be positive for one or more of the pathogens tested. The overall positivity for VZV, HSV, CMV and T. gondii was found to be 16 (12.7%), 7 (5.6%), 5 (3.9%), and 9 (7.1%); with mean pathogen load of 5.07×10, 9.5×10, 1.08×10 and 394 (copies/μl) respectively. The development of highly sensitive and specific assay for early differentiation of pathogens is important for the early initiation of treatment thereby preventing irreversible damage to the ocular structures.