The local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA used to identify skin sensitizers. This assay measures the proliferation of auricular lymph node cells (LNCs) during the induction phase of skin sensitization and the number of BrdU-positive LNCs using flow cytometry. We determined if LLNA: BrdU-FCM can evaluate the skin sensitization potential of 20 substances, including 16 sensitizers and 4 non-sensitizers, that were tested using LLNA: DA and LLNA: BrdU-ELISA but not listed in OECD TG 429. After selecting appropriate vehicles and conducting pre-screen tests in 2 phases, solvents and test concentrations for the main test were determined. In the main study, we measured changes in LN weight, the number of LNCs, and the proportion of BrdU incorporated into LNCs to calculate stimulation indexes (SI). SI was calculated based on the total number of LNCs and BrdU incorporation in LNCs. We found that all substances were correctly classified as sensitizers or non-sensitizers. Overall, we confirmed that the LLNA: BrdU-FCM can evaluate skin sensitization potential of the 20 substances. Additionally, our results of combining 22 reference substances listed in OECD TG 429 and 20 additional substances showed that concordance of LLNA: BrdU-FCM with the LLNA was higher than before.