Knowledge of virus and replication kinetic is one of the most important issues in the vaccine production. The present study aimed to evaluate the best harvesting time of H9N2 avian influenza virus (AIV) vaccine strain inoculated in specific pathogen free (SPF) embryonated chicken eggs (ECE)s. For this purpose, 10-5 dilution of AIV (A/Chicken/Iran/99/H9N2) was inoculated into 336, 11-day old ECEs at the rate of 0.1 ml/ECE via intera-allantoic. Amnio-allantoic fluid (AAF) and chorioalantoic membranes were collected at 2 hours intervals up to 96 post inoculations from 7 eggs in each trial. The presence of virus in the harvested suspensions was evaluated by Hemagglutination assay (HA) and Egg infective dose 50 (EID50) assays. Our results showed that, the best virus harvesting time for vaccine production is between 50 – 60 hours post inoculation (hpi) in ECEs. This finding may provide possibility of the sufficient and increasing rate of production, immunogenicity and economic factors in H9N2 influenza vaccine production.