This work aimed to evaluate the effects of in ovo vaccination procedures on incubation of Japanese quail eggs. The experiment was carried out in a (4 x 3) factorial design with 12 experimental treatments (4 injection days x 3 injection protocols). The injections were tested on four incubation days: at 0, 5, 10 or 15. On each injection day, the eggs were submitted to one out of three distinct injection procedures: saline injection and Newcastle disease (ND) vaccine plus saline or industrial diluent. The eggs were incubated at 37.5º C and 60% RH. All eggs and hatched quails were weighed. Unhatched eggs were opened to classify embryo mortality. Hatched quails were raised to obtain blood to evaluate antibody response against Newcastle disease virus (NDV). The injection process itself (saline) was not harmful at 10 and 15 days of incubation for Japanese quail eggs; however, in ovo vaccination with live ND vaccine (HB1 strain) is not recommended to fertile quail eggs at any incubation periods due to high levels of embryo mortality and poor post-hatch antibody titers.