Avian viral problems have been consistently reported in commercial poultry of Pakistan causing heavy economic losses to the poultry farmers. Authentic idenfication and confirmation of the causative agent is always been question mark for the selection of vaccinal strain in this regard. Current study was therefore undertaken to optimize the virus neutralization test for the serological survey of vaccinated poultry particularly for avian influenza virus’s subtypes and Newcastle disease virus. Various physiochemical factors such as concentration of antigen and antibody, Incubation temperature and incubation period for in vitro and in-vivo reaction of antigen and antibody were optimized in chicken embryonated eggs