In this report, the data are presented on the use of synthetic biotinylated oligosaccharides corresponding to different fragments of capsular polysaccharides (CP) from S. pneumoniae serotypes 3 and 14 for evaluation of the level of vaccine-induced antibodies in the serum of mice immunized with conjugates of oligosaccharide ligands with bovine sera albumin (BSA). The level of antigen-specific antibodies assessed by enzyme-linked immunosorbent assay (ELISA) using biotinylated oligosaccharides immobilized on the surface of streptavidin precoated ELISA plates was substantially higher than that assessed using conventional ELISA plates with the bacterial CP as a coating antigen. Hence, the ELISA protocol using biotinylated oligosaccharides is much more sensitive as compared to the conventional protocol, and it can be used for the detection of antibodies in highly diluted antisera. The highest antibody titer against biotinylated oligosaccharides was detected in the antisera of mice immunized with the conjugates of BSA with tetrasaccharide ligands related to fragments of CP of two pneumococcal serotypes under study. The antigen-binding ability of antibodies to tetrasaccharides related to one and two repeating units of CP from S. pneumoniae types 14 and 3, respectively, was higher than that of antibodies to BSA conjugated with oligosaccharides of other chemical structure in the reaction of biotinylated oligosaccharides and in the bacterial agglutination test with live cells on the glass slide but not in the inhibition of ELISA with bacterial CP. This research provided a rationale for the choice of tetrasaccharide ligands for the design of the third generation vaccines against S. pneumoniae types 3 and 14 and showed the validity of the ELISA protocol with biotinylated oligosaccharides as coating antigens for the assessment of the level of neoglycoconjugate-induced antibodies.