Eukaryotic pre-mRNA splicing is an essential step in gene expression for all genes that contain introns. In contrast with transcription and translation, few well-characterized chemical inhibitors are available with which to dissect the splicing process, particularly in cells. Therefore, the identification of specific, small molecules that either inhibit, or modify, pre-mRNA splicing would be valuable for research and potentially also for therapeutic applications. We have screened a highly curated library of 71,504 drug-like small molecules using a high throughput in vitro splicing assay. This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. One of these splicing modulators, DDD00107587 (termed "madrasin" i.e., (2-((7methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one RNA splicing inhibitor), was studied in more detail. Madrasin interferes with the early stages of spliceosome assembly and stalls spliceosome assembly at the A complex. Madrasin is cytotoxic at higher concentrations, while at lower concentrations it induces cell cycle arrest, promotes a specific reorganization of subnuclear protein localization and modulates splicing of multiple pre-mRNAs in both HeLa and HEK293 cells.