AbstractConstitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell‐based assays using synthetic TCF/LEF (T‐cell factor/lymphoid enhancer factor) reporters, as readouts of β‐catenin/TCF‐dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia‐lyase gene (HAL) that was negatively regulated by β‐catenin/TCF‐dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β‐catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β‐catenin signaling pathway. The utility of our system could be expanded to examine other disease‐associated pathways beyond the Wnt/β‐catenin signaling pathway.