Current diagnosis of Acute Rheumatic Fever and Rheumatic Heart Disease (RHD) utilises a combination of clinical observation, indirect laboratory tests, and echocardiography. The application of these criteria remains a major challenge where ARF/RHD are often most prevalent. A simple non-ambiguous diagnostic test for ARF that is easily deployable remains a highly desirable yet elusive goal with the potential to result in faster interventions. We previously reported the use of peptide array technology aligned with the rat autoimmune valvulitis model as a tool for the identification of candidate peptide sequences from the M-protein that could for the basis of a simple serological based ARF diagnostic. Here we evaluated the immune responses to three peptides (p24, p24a.1 and p20a.1) arising from these prior studies.  For these assays human ARF and control sera from Australia or Aotearoa were used in ELISA against biotinylated peptides attached to streptavidin coated plates. Our findings show an increase in mean absorbance for ARF sera for all three peptides when compared to sera from healthy controls in both panels of sera. However no statistical differences were observed between the New Zealand ARF sera and sera collected from patients with streptococcal bacteraemia.