Vaccination programs for the control of bluetongue (BT) in ruminants have limitations due to difficulties in differentiating infected from vaccinated animals (DIVA). To overcome this problem a DIVA test that looks at a differential immune response to bluetongue virus (BTV) non-structural protein 3 (NS3) was developed. The NS3 encoding gene of strain BTV4/22045/PT04 was inserted into expression vector pET-28a and expressed in Escherichia coli strain JM109. Recombinant NS3 protein was used as an antigen in an indirect ELISA (NS3-ELISA) to measure the serologic response to NS3 protein in cattle and sheep. Following a cattle vaccination/challenge experiment with a bivalent inactivated BTV 2-4 vaccine, NS3 antibodies were detected at approximately 15 days post-infection in control unvaccinated animal, while vaccinated animals did not develop detectable NS3 antibodies and, with exception of one, remained negative even after virus challenge. The inactivated vaccine induced antibodies against the major core structural protein (VP7) of BTV as well as neutralizing antibodies in all animals. To evaluate the applicability of NS3-ELISA in field scenario, a total of 562 serum samples collected from uninfected, BTV-infected and vaccinated animals were tested for NS3 antibodies. Taken together, the results confirm that NS3 antibodies were induced to the greatest levels in animals infected with BTV in comparison to the levels induced in animals immunized with inactivated BTV vaccines, implying that antibody response to NS3 allows the differentiation between infected and vaccinated animals.