Human Herpes Simplex Viruses 1 and 2 (HSV-1 and HSV-2) are large genome DNA viruses that establish a persistent infection in sensory neurons and commonly manifest as recurring oral or genital erosions. Major infection is usually acquired in childhood and is most often asymptomatic, subsequent to which virus becomes latent in neurons of cranial nerve ganglia (HSV-1) or sacral ganglia (HSV-2). The present study aimed at the preliminary screening of suspected HSV encephalitis cases. A total of 100 cerebrospinal fluid (CSF) specimens were collected from clinically suspected HSV encephalitis cases representing various government and private hospitals of Chennai and were subjected to molecular detection by nested real-time Polymerase Chain Reaction (PCR) at the Virology Laboratory, King Institute of Preventive Medicine and Research (KIPM & R). The detection of HSV glycoprotein gene confirmed a total of 15 (15% of 100) samples as positive for HSV infection by PCR. In addition, three cases were also confirmed to be positive based on their cultivation in vero cell line.