Swine influenza continues to be an economically significant respiratory disease in pigs of all ages. Serological testing is commonly employed to detect animals that have been exposed to swine influenza virus (SIV) because the disease has a relatively short course and the causative agent becomes quickly undetectable in infected animals. Serology for SIV is also used to assess the immune status of pigs at various stages within an operation so that the level of herd immunity or timing of vaccination can be determined. However, as the performance of serologic assays commonly used, such as hemagglutination-inhibition (HI) test and commercial ELISA (IDEXX), is restricted to the subtype of virus, the diagnostic value of serologic testing has been diminished by the emergence of new subtypes and antigenic drift within the same subtype, and serodiagnosis is often confounded by the presence of antibody conferred by vaccination. Such shortcomings of current SIV serology will continue as genetic and antigenic evolution is one of the biological characteristics of the virus. The problems emphasize the need for a subtype-unrestricted serodiagnostic test (i.e., better sensitivity), which can also differentiate naturally infected animals from vaccinated animals (i.e., better specificity). A recent study conducted in our laboratory revealed potential antigen candidates (i.e., nucleoprotein and NS1 protein) to fulfill the need. The present study put these 2 antigens together into a serologic assay in an ELISA format and demonstrated its utility in detecting pigs exposed to SIV regardless of its subtype and differentiating vaccine-induced antibody from natural infection (i.e., DIVA test). The availability of such a reliable assay with DIVA capability in diagnostic laboratories is expected to be a benefit to the swine industry since swine herds and pig populations can be consistently and reliably monitored for swine influenza due to existing or emerging SIV. In addition, the availability of such a test will be of great assistance in determining the time for prophylaxis.