Background: Exposure to ionizing radiation is known to induce oxidative stress followed by damage to critical biomolecules like lipids, proteins and DNA through radiolysis of cellular water. Since radiation has been widely used as an important tool in therapy of cancer, the detailed investigation regarding the DNA damage and repair kinetics would help to predict the radiation sensitivity of cells. The present study is focused on quantification of DNA damage and repair kinetics of human peripheral blood lymphocytes after 8 MeV pulsed electron beam irradiation in vitro. Materials and Methods: DNA damage and repair kinetics in human blood cells were studied using alkaline comet assay. The dose-response curves for the dose range of 0-5 Gy were established using 8 MeV electron beam. Repair kinetics was studied by incubating the cells from 0 to 90 min at 37°C after irradiation. For quantification of DNA damage, percentage Tail DNA, Tail length, Tail moment and Olive Tail moment were used as a comet parameter. Results and Conclusion: The study reveals that, induction of DNA damage by 8 MeV pulsed electron beam is near linear with a negligible negative quadratic component at high doses. This small quadratic component is attributed to high dose rate during the pulsed irradiation. The DNA repair halftime and mean repair time for human blood lymphocytes were found to be varying between 9.29 - 23.78 min and 13.41 - 34.31 min respectively. The repair rate is found to be maximum in initial 15 minutes and almost constant after 60 min.