Background and Aim We need the next-generation of whole-inactivated influenza vaccines to create stronger cross-protection against different influenza subtypes. This research aimed to apply the inactivation process of the influenza virus through gamma radiation as a candidate for the development of whole-inactivated vaccines. Methods and Materials The influenza virus strain A/PR/8/34 (A/Puerto Rico/8/34 [H1N1]) was propagated in Madin-Darby Canine Kidney (MDCK) epithelial cells. After ultrafiltration, the virus infectivity titer was calculated by 50% Tissue Culture Infectious Dose (TCID 50%) method based on the Karber formula. Alternatively, the gamma cell-220 was applied for virus inactivation via gamma rays. The D10 value factor and optimum dose of virus inactivation were calculated based on the dose/survival curve and the initial viral titer. In addition, antigenic properties of irradiated viruses compared to un-irradiated viruses and complete inactivation of the irradiated samples with optimum dose were also evaluated by hemagglutination assay and safety test, respectively.