Human Cytomegaloviruses and Herpes Simplex Viruses are the major cause of serious viral complications in pregnant women. Conventional screening methods that is ELISA for detecting Human Cytomegalovirus (HCMV) and Herpes Simplex virus (HSV) tend to be slow and insensitive. Therefore in this work, a rapid Real Time PCR-based assay was designed to detect CMV and HSV which are responsible for causing various viral infections among pregnant women in Khyber Pakhtunkhwa. The present study aimed to compare the specificity and sensitivity of the PCR-based assay with ELISA based assay in the diagnosis of HCMV and HSV infections in pregnant women in Khyber PakhtunKhwa. In order to check the validity of Real time PCR technique in the early diagnosis of the infection, serologicalresults were compared to the results of Real Time PCR based detection of HCMV and HSV from serum samples. Our study revealed that among the ELISA screened 175 positive sera samples i.e 81 (70%) of CMV and 34(30%) of HSV, while 17 (10%) had positive results for CMV DNA and 7(4%) had HSV DNA through real-time PCR. Real time PCR was more sensitive and reliable method in diagnosis of CMV and HSV infections in pregnant women in this comparative study.