Dabrafenib and irinotecan are two drugs that can be utilized to treat melanoma. A previous in vivo study has shown that dabrafenib enhances the antitumor activity of irinotecan in a xenograft model with unclear mechanism. This study aims to investigate the inhibition of dabrafenib on SN-38 (the active metabolite of irinotecan) glucuronidation using human liver microsomes (HLMs) and recombinant human UGT1A1, trying to elucidate the possible mechanism underlying the synergistic effect. Our data indicated that dabrafenib noncompetitively inhibited SN-38 glucuronidation in pooled HLMs and recombinant UGT1A1 with a K i,u value was 12.43 ± 0.28 and 2.64 ± 0.27 μM, respectively. Based on the in vitro K i,u value and estimation of kinetic parameters, dabrafenib administered at 150 mg twice daily may result in about 17%-81.9% increase in the area under the curve (AUC) of SN-38 in vivo . Moreover, the ratios of [ I ] gut / K i,u are 0.70 and 3.32 in HLMs and recombinant UGT1A1, respectively, indicating a high risk of drug-drug interactions (DDIs) when dabrafenib was used in combination with irinotecan. Our study provides a basis for further development and optimization of this combination in clinical research.