We applied a one-step 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). The cold-adapted reassortant influenza viruses A/H1N1 pdm09, A/H3N2, B/Yamagata and B/Victoria, included in the composition of the LAIV in 2015-2016, 2017-2018 and 2018-2019 flu season were studied for reproductive activity in MDCK cells as part of a mono-vaccine and tri-vaccine. For this we have developed a set of specific primers and probes. Method validation was performed using ELISA-test after mouse monoclonal antibodies to hemagglutinin (HA) staining of MDCK monolayer. Influenza B viruses B/Yamagata and B/Victoria were studied in MDCK cells in mono-infection and coinfection with different multiplicity of infection (MOI) using quantitative rRT-PCR. • RT-PCR analysis was adjusted to assess the growth characteristics of cold-adapted reassortant influenza viruses in MDCK cell line. The greatest suppression in the composition of the tri-vaccine was exposed to the H1N1 pdm09 LAIV component. • Influenza B viruses are least suppressed in trivalent LAIV. Influenza viruses B/Yamagata and B/Victoria reproduced as part of a mixed preparation not lower, if not better than as a mono-preparation at an MOI of 0.1. At an MOI of 0.01, the reproduction of both B/Yamagata and B/Victoria in the mixture was reduced compared to mono-vaccine. • The interference of trivalent LAIV vaccine viruses in MDCK cells was minimal at low dilutions. This indicates that it is undesirable to reduce the titers of vaccine viruses, including at the stages of transportation and storage of LAIV