The development of a discriminatory test, based on the differentiation between N1 and N3 antibodies, to be used in the framework of a vaccination program, based on vaccination with a heterologous H7N3 inactivated vaccine against the Italian H7N1 field virus, is reported. The indirect immunofluorescence antibody (iIFA) assay was based on the expression of the N1 protein in a baculovirus system. HighFive® insect cells were transfected with the recombinant virus and used as an antigen in the iIFA test. Preliminary validation on 608 turkey sera yielded relative sensitivity and specificity of 98.1% and 95.7%, respectively, when compared to the HI test with an almost perfect agreement between the two methods (Kappa value = 0.93). It is concluded that the iIFA test is a valid tool for monitoring avian influenza infection in a vaccinated population.