The effective control of foot-and-mouth disease (FMD) requires sensitive, specific and rapid diagnostic tools. However, the control and eradication of FMD in Africa is complicated by, among other factors, the existence of five of the seven FMD virus (FMDV) serotypes, including the SAT-serotypes 1, 2 and 3 that are genetically and antigenically the most variable FMDV serotypes. A key diagnostic assay to enable a country to re-gain its FMD-free status and for FMD surveillance, is the 3ABC or the non-structural protein (NSP) enzyme-linked immunosorbent assay (ELISA). Although many kits are available to detect 3ABC antibodies, none has been developed specifically for the variable SAT serotypes. This study designed a SAT-specific NSP ELISA and determined whether this assay could better detect NSP-specific antibodies from FMDV SAT-infected livestock. The assay’s performance was compared to validated NSP assays (PrioCheck®-NSP and IZSLER-NSP), using panels of field and experimental sera, vaccinated and/or infected with FMDV SAT1, SAT2 or SAT3. The sensitivity () of the SAT-NSP was estimated as 76% (70%, 81%) whereas the specificity was 96% (95%, 98%) at a 95% confidence interval. The sensitivity and specificity were comparable to the commercial NSP assays, PrioCheck®-NSP (82% and 99%, respectively) and IZSLER-NSP (78% and 98%, respectively). Good correlations were observed for all three assays.