Herpes simplex viruses (HSV) are important human pathogens causing diseases in a variety of different human tissues and animal species. HSV-1 is being most often transmitted non-sexually and HSV-2 most usually transmitted sexually. Primary HSV infections are usually symptomatic but may be sub clinical. Worldwide 65% - 90% rate of HSV infection found in different part of world and HSV1 is more common than HSV2. Few studies from India documented for HSV 1 and HSV 2 infections in different population groups. In India HSV 1 and HSV 2 seroprevalence in adults are 63% and 16.6% respectively. Due to the high impact of these viruses on our health, there should be a rapid, sensitive, specific and cost effective method for the detection of these viruses. Presently Herpes viruses are diagnosis by virus isolation, rapid commercial kits, conventional and real time PCR. Virus isolation is time consuming, rapid kit have low sensitivity and specificity while real time PCR are costly hence convention PCR are comparatively good choice. Thus this study was conceptualized for HSV1 and HSV2 detection by conventional PCR from standard strains of HSV1 and HSV2 which can on the clinical samples. First we standardized two monoplex PCR in two separate PCR tubes for HSV1 and HSV2 then Single tube Multiplex PCR for HSV 1 and HSV 2 was standardized. The specificity of the primers was checked with other related virus such as human cytomegalovirus. Multiplex PCR assay was applied on panel of clinical samples. The study will serve as rapid, accurate, specific and sensitive diagnostic assay which is crucial for HSV1 and HSV2 to help in patient management and prevention of spread of disease.