Blood transfusion is most important part of medical treatment and is also associated with risk of transfusion transmitted infections (TTIs). Hence screening of blood bags is important to ensure safe blood transfusion. The present study was done to evaluate the ability of nucleic acid testing (NAT) and to compare it with ELISA assay to detect Window period for HBV, HCV, and HIV in the donor population of the blood bank of Karwar institute of Medical sciences, Karwar, Karnataka. A total of 3183 donors were screened over 4 ½ years to assess the seroprevalence of infectious disease markers. Blood units were screened for the five commonest TTIs namely HIV I & II, HBsAg, HCV syphilis and malaria using screening test like ELISA and Rapid Kit, peripheral smear and VDRL tests. All reactive sample were retested. Seropositive blood bags were discarded. All non-reactive samples were sent for confirmation testing by NAT technique for HIV I & II, HBsAg, HCV to reduce the risk of TTIs in the recipients, thus providing an additional layer of blood safety. Out of 3183 blood donors, 17 were seropositive for the TTIs. Totally 14 units were positive for HBV infection, out of those 13 were detected HbsAg positive by ELISA and 1 seropositive donor which was not detected by the serological test was detected by the NAT. NAT implementation is likely to reduce the TTIs and its implementation will be a valuable addition to the existing safety efforts.