Survivin, a key member of the chromatin passenger complex (CPC), is often highly expressed in human cancers, making it a promising target for cancer treatment. Out of the numerous reported Survivin inhibitors, YM155 is only one entering clinical trial, but was recently failed in the Phase II trial. It is important to develop Survivin inhibitors with new strategies. We recently reported that both Survivin and its binding protein Borealin in the CPC complex are essential for the development of hepatocellular carcinoma, suggesting that disrupting the interaction between Survivin and Borealin would be a promising strategy. Here, we developed a high-throughput screening method based on bimolecular fluorescence complementation (BiFC) technology in cultured cells, which allowed the identification of small chemical inhibitors specifically blocking the Survivin and Borealin interaction. Primary hits from BiFC were further validated in an in vitro AlphaScreen system, which detects the direct interactions of Survivin and Borealin. Etoposide was identified as one of the effective hits. Direct interaction between Survivin and Etoposide was confirmed by surface plasmon resonance assay, and molecular docking analysis suggested the structural information on how Etoposide inhibits the Survivin and Borealin interaction. These results demonstrate a screening system to identify small molecule chemicals inhibiting Survivin and Borealin interaction. In future, an even larger scale screening may lead to identification of better Survivin and Borealin inhibitors. (C) 2017 Elsevier Inc. All rights reserved.