Herpes simplex virus (HSV) keratitis is a leading cause of vision impairment worldwide; however, early diagnosis and timely treatment has good prognosis. Currently, there are no set diagnostic criteria, and most ophthalmologists depend on characteristic clinical findings and history for diagnosis. The varying and often atypical presentations of HSV keratitis necessitate the use of alternative laboratory diagnostic methods for an accurate diagnosis and exclusion of other infective etiologies. Viral culture, the “gold standard” for HSV diagnosis, has largely been replaced by polymerase chain reaction (PCR). Real-time PCR has high sensitivity and specificity for HSV detection in both corneal scrapings and tear samples. Other more rapid but less sensitive methods include immunofluorescent assay (IFA), ELISA for antibody detection and cytologic examination. Novel, yet less explored, techniques like the multiplex dot hybridization (MDH) assay, ICP0 protein detection in tears, and immunochromatographic assays (ICGA) for both the detection of HSV-DNA (AmpliVue) and glycoprotein D antigens may be used as adjuncts or preliminary diagnostic tools in clinical settings.