The antigenic characterisation of three H3N2 type A influenza strains by mass spectrometry is described. The approach, developed in this laboratory, employs matrix-assisted laser desorption ionisation (MALDI) mass spectrometry to analyse gel-resolved antigens, post their proteolysis and treatment with monoclonal antibodies. The primary structure and antigenicity of the component antigens of the virus can be determined in a single step. Four antigenic domains of hemagglutinin have been identified and these are localised at residues 109-125, 158-170 and 316-326 of the HA1 subunit and to residues 159-183 of the HA2 subunit. The results demonstrate the applicability of the approach for identifying antigenic determinants across various H3N2 strains with high throughput and at low sample levels. Comparative rates of antibody binding between two of the antigenic peptides have also been reported.