The trans-sialidase (TS), a virulence factor expressed on the surface of Trypanosoma cruzi, the agent of Chagas disease, is an enzyme that transfers sialic acids between glycoconjugates. In humans and most tested mammals, the onset of the chronic phase of T. cruzi infection correlates with the elicitation of antibodies directed to the TS catalytic domain, which inhibit the sialyl residues transfer reaction in vitro and in vivo. The method described here, termed trans-sialidase inhibition assay (TIA), enables the detection of TS-neutralizing antibodies in serum samples of different mammalian species, without the use of conjugated secondary reagents. The high specificity and exquisite sensitivity displayed by the TIA allow to overcome the limitations of routinely used Chagas disease serodiagnostic assays.