Infectious uveitis is a serious condition requiring rapid diagnosis and therapeutic management. Molecular tools, such as PCR and its variants, have significantly changed the diagnostic approach during the last 10 years. Presumed and empirical diagnosis should be excluded in the face of atypical clinical presentations, but also inadequate response to anti-inflammatory drugs. It is of particular importance to note that most of these tests need to be processed in specialized central laboratories to ensure the best sensitivity and specificity. Molecular techniques have their own limits, sometimes caused by inadequate primers or PCR-inhibitors and contaminants. PCR positivity means detection of pathogen DNA, but does not confirm a productive infection. Cultures from ocular fluids should be improved to obtain complementary data about pathogen life cycles and resistance to antibiotics or antivirals. Furthermore, diagnostic yield is significantly increased when PCR and local antibody production are associated, especially in viral infections. This step is of particular importance to identify new infectious entities in cases that are presently considered to be idiopathic or autoimmune disorders.