A procedure for the determination of activity and linkage specificity of sialidases is described. The sialoglycoprotein fetuin is coated onto a microtiter plate and incubated with sialidases from different sources. Enzymatic activities and linkage specificities are then determined by a sandwich method which measured the binding of different lectins to fetuin. The lectins used were peanut agglutinin (PNA) from Arachis hypogaea, which binds specifically the galactose beta-1-3-N-acetylgalactosamine structures that are unmasked following sialidase treatment of fetuin, the lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA) that are specific for alpha-2-6 and alpha-2-3 bound sialic acids, respectively, and the slug agglutinin from Limax flavus (LFA) that is specific for N-acetyl and N-glycolyl neuraminic acids. Increased PNA and decreased LFA, SNA, and MAA lectin binding correlated with sialidase-induced desialylation of the substrate. In this report, the assay was used to determine the activities and specificities of influenza, Vibrio cholerae, and Arthrobacter ureafaciens sialidases.