The detection of anti-Streptococcus equi antibodies in the blood serum of horses can assist with the identification of apparently healthy persistently infected carriers and the prevention of strangles outbreaks. The aim of the current study was to use genome sequencing data to develop an indirect enzyme linked immunosorbent assay (iELISA) that targets two S. equi-specific protein fragments. The sensitivity and specificity of the antigen A and antigen C iELISAs were compared to an SeM-based iELISA marketed by IDvet - diagnostic Vétérinaire (IDvet). Individually, each assay compromised specificity in order to achieve sufficient sensitivity (SeM iELISA had a sensitivity of 89.9%, but a specificity of only 77.0%) or sensitivity to achieve high specificity. However, combining the results of the antigen A and antigen C iELISAs permitted optimisation of both sensitivity (93.3%) and specificity (99.3%), providing a robust assay for the identification of horses exposed to S. equi.