To prepare hemagglutination inhibition (HI) antigen the egg drop syndrome virus (EDSV) was propagated in 10-day-old embryonated duck eggs. The virus antigen was inactivated with two methods including heating (65°C) and adding 0.5% formaldehyde by considering low destroying effect on hemagglutination (HA) and HI titers. The EDSV HI titers of 310 sera and 100 yolks obtained from 23 chicken farms and specific pathogen free (SPF) chickens before and after EDS vaccination, in various ages were evaluated. Specificity, HA activity, stability and electron microscopic observations were similar in the prepared and standard antigens. The HI titers showed an equal sensitivity and specificity and, complete similarity (>99%) between the results obtained by prepared and standard antigens. Detection of antibody against EDSV using HI test showed slightly less sensitivity but more specificity compared with ELISA test. Upon the results it is suggested that inactivated EDS antigen prepared by either heat or formalin treatment could successfully be used in avian diagnosis laboratories for routine flocks monitoring, EDS vaccine controlling and also surveillance of EDS vaccination program in poultry industry.