Influenza virus H9 was standardized for hemagglutination assay using different factors such as red blood cell (RBC) types, concentrations, diluent types, and storage times. Avian influenza virus H9 was grown in embryonated chicken eggs and confirmed by spot agglutination. A significant (P < 0.05) difference with the highest mean titer (9.00 ± 0.00) was observed using RBCs from different species. Nonsignificant differences (P > 0.05) were found between human blood type O, chicken, and dog RBCs, as well as among rabbit, pigeon, sheep, and parrot. The highest titer (9.00 ± 0.00) with a nonsignificant difference was found using virus stored at 4 °C and -20 °C while 37 °C showed the lowest significant mean hemagglutinin (HA) titer (11.08 ± 188.21). Nonsignificant differences were observed in HA titers against H9 virus stored for 3, 4, 5, and 6 months. Nonsignificant differences were found between the use of normal saline and 0.5% peptone water with the lowest HA titers of 7.83 ± 0.40 and 8.00 ± 0.00, respectively, while the highest HA titer (9.00 ± 0.00) with nonsignificant difference was observed by using HA-HI buffer and phosphate buffer saline as diluents. RBCs with 0.5% and 1% concentrations showed nonsignificant difference in HA titer but significant difference with 0.1% RBCs.