PURPOSE: To report the novel application of nontreponemal and treponemal antibody to confirm diagnosis of ocular syphilis from vitreous samples. METHODS: Two distinct case reports emphasizing the importance of confirmatory vitreous treponemal antibody. Multimodal imaging of patients was also applied. RESULTS: We report two distinct cases with positive serum treponemal antibody but opposing vitreous treponemal antibody results. One case with a positive vitreous test responded well to antisyphilitic treatment. By contrast, a case with a negative vitreous result was changed to serpiginous choroiditis, eventually cured by immunomodulatory treatment. CONCLUSION: Intraocular fluid analysis of nontreponemal and treponemal antibody may play an important role in ruling out suspected ocular syphilis in settings without a polymerase chain reaction facility, especially immunocompromised patients who are at risk of multiple infections. Further studies are needed to establish the sensitivity and specificity of nontreponemal and treponemal antibody test on vitreous samples.