Salmonella enterica subsp. enterica is a major pathogen that causes zoonotic foodborne diseases worldwide. Some Salmonella serovars possess two antigenic phases for flagellin: phase 1 and 2. In Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellin is antigenically divided into "Hi" as phase 1 and "H1 or H2" as phase 2. Flagellin phase variation is regulated by inversion of hin gene. We focused on the inversion of hin and developed a real-time PCR system to quantitatively measure the proportion of bacterial cells expressing each phase of flagellin. In this study, we demonstrated that our newly developed real-time PCR system shows high quantitative accuracy and aligns with flagellin expression status. Furthermore, the newly developed real-time PCR system was applicable to various S. Typhimurium laboratory and field strains. This newly developed real-time PCR system has the potential to become a powerful tool for analyzing flagellin phase variation.