Among the avian influenza virus (AIV) subtypes, H9N2 virus has the potential to cause an avian influenza (AI) pandemic and vaccination with inactivated vaccines to prevent or reduce losses due to infections is common. Formalin is one of the most important inactivators in AIV inactivated vaccine production process and incorrect inactivation may be due to secondary irreparable problems. So we evaluate these conditions to optimize inactivation factors such as temperature, time, shaking and formalin concentration. For the optimization of factors, we used different treatment concentrations (0.05, 0.075, 0.1, 0.125, 0.15, and 0.2%), time (12, 16 and 21 hour), temperatures (25, 30, 35, 37 and 39oC) and shaking condition. The titer of inactivated viruses was evaluated with standard hemaglutination (HA) test and non infectivity of those was assessed with inactivation test (IT). The optimum condition that could inactivate the infectivity of AIV (H9N2) was 0.05% concentration in 12 h and 37oC. The shaking did not have any significant effects on inactivation process. So the mentioned qualifications are the optimum condition for virus inactivation in the production of inactivated AI vaccine process.