Background: The objective of the present study was to compare reproduction of trivalent LAIV vaccine strains in MDCK cells and to perform quantitative RT-PCR analysis of trivalent LAIV replication after inoculation in mice. Methods: We applied a reverse transcriptase real-time PCR (rRT-PCR) analysis using TaqMan technique to evaluate the infectious titers of vaccine strains containing in trivalent live influenza vaccines (LAIVs). We confirmed the PCR data in ELISA using staining of MDCK monolayer with mouse monoclonal antibodies to hemagglutinin. Results: The viral load during the reproduction of mono-vaccines and trivalent LAIV in MDCK cells was similar at low dilutions. The content of vaccine viruses was evaluated using quantitative RT-PCR analysis in the nasal turbinate and lungs of CBA mice on day 3 after intranasal immunization. It was shown that despite the almost complete absence of reproduction of the A/H3N2 virus in mice, the immune response of A/H3N2-specific antibodies was formed at the same level as to other viruses. In MDCK cells, a decreased infectious titers of vaccine viruses in trivalent LAIV compared to mono-vaccines was demonstrated except for B/Yamagata virus. Conclusion: RT-PCR analysis is applicable to assess the growth characteristics of cold-adapted reassortant influenza viruses in vitro and in mice. The interference of trivalent LAIV vaccine viruses in MDCK cells was minimal at low dilutions. In mice, decrease in infectious titers did not lead to a decline of the immunogenicity.