Abstract Recent progress in understanding the molecular mechanisms that underlie bladder cancer carcinogenesis offers the prospect of specifically targeting signaling pathways to achieve more effective and rational treatment. In a novel, 3-dimensional high-throughput drug screening (3D HTS) platform to identify novel agents affecting bladder cancer cells, 24 molecularly-targeted agents were screened in seven human bladder cancer cell lines (UCUM3, T24, SW780, RT4, J82, 5637 and 253J-BV). The efficacy of 24 targeted agents on the bladder cell lines varied dramatically on their genomic alterations. BEZ235 (mTOR and PI3K inhibitor) showed antitumor effect in most of cell lines except UMUC3. Another mTOR inhibitor, AZD2014 (inhibitors of mTORC1 and mTORC2) also had IC50 <2 μM in 3 cell lines (5637, J82 and RT4). However, FGFR targeted agents (AZD4547 and BGJ398) did not show the efficacy in cell lines with FGFR3 alterations (J82, SW780 and RT4). In HER2 mutated/amplified cell line (5637), dacomitinib, lapatinib and neratinib showed anti-tumor effect. Immunoblot assay showed that lapatinib and neratinib suppressed phosphorylated HER2 and downstream molecules such as p-AKT or p-ERK. In conclusion, 3D HTS drug screening platform could be a useful preclinical tool to evaluate various kinds of drugs rapidly, and eventually provide a valuable insight into precision medicine along with comprehensive genomic analysis. Note: This abstract was not presented at the meeting. Citation Format: Sang-Cheol Lee, Young Saing Kim, In Gyu Hwang, Su Jin Lee, Se Hoon Park. Three-dimensional high-throughput drug screening for bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 997.