Background: The production of intraocular antibodies is considered a specific marker for active infectious uveitis. The aim of our study was to evaluate the diagnostic value of aqueous humor analysis in consecutive patients referred to a tertiary clinical center.  Methods: We analyzed 91 paired aqueous humor/serum samples from 89 patients with intraocular inflammation. In 71 patients aqueous humor analysis was used as a positive or negative confirmation of the suspected cause, whereas in 18 patients the clinical diagnosis was completely uncertain. A modified micro-ELISA technique was used to detect intraocular IgG production against Toxoplasma gondii, varicella zoster virus, herpes simplex virus and cytomegalovirus. Statistical analysis was performed using the ”Cohen’s kappa” test.Results: Specific intra-ocular antibody production could be detected in 12 (66.7%) of 18 patients with uncertain diagnosis. Subsequently initiated therapy led to clinical improvement in 10 patients, whereas 2 patients remained unchanged. In 2 (2.8%) of 71 patients aqueous humor analysis led to revision of the initially suspected etiology and to a change of therapy. Statistical analysis showed a significant accordance of diagnosis and aqueous humor analysis (P<0.01).  Conclusion: In patients with infectious uveitis, analysis of intraocular synthesis of specific antibodies is a valuable tool to establish the etiology rapidly and allows initiation of targeted antimicrobial treatment.