Foot-and-mouth disease (FMD) is a highly contagious viral disease of transboundary importance. In India, since the launch of the FMD control programme, there has been a substantial increase in the vaccinated bovine population. In this scenario, there is a need for additional locally developed non-structural protein (NSP)-based immnoassays for efficient identification of FMD virus (FMDV)-infected animals in the vaccinated population. The 2B NSP of FMDV, lacking the transmembrane domain (Delta 2B), was expressed successfully in a prokaryotic system, and an indirect ELISA (I-ELISA) was developed and validated in this study. The diagnostic sensitivity and specificity of the Delta 2B I-ELISA were found to be 95.3 % and 94.6 %, respectively. In experimentally infected cattle, the assay could consistently detect Delta 2B-NSP-specific antibodies from 10 to approximately 400 days postinfection. The assay was further validated with bovine serum samples collected randomly from different parts of the country. The performance of the Delta 2B I-ELISA was compared with the in-house r3AB3 I-ELISA, and the overall concordance in test results was found to be 86.49 %. The Delta 2B I-ELISA could be useful as a screening or confirmatory assay in the surveillance of FMD irrespective of vaccination.