The spliceosome catalyzes pre-messenger RNA (pre-mRNA) splicing, an essential process in eukaryotic gene expression in which non-protein-coding sequences are removed from pre-mRNA. The spliceosome is a large, molecular complex composed of five small nuclear RNAs (snRNAs) and over 100 proteins. Large-scale rearrangements of the snRNAs and their associated proteins, including changes in base-pairing partners, are required to properly identify the intron-containing pre-mRNA, position it within the spliceosome, and complete the cleavage and ligation reactions of splicing. Despite detailed knowledge of the composition of the spliceosome at various stages of assembly, the critical signals and conformational changes that drive the dynamic rearrangements required for pre-mRNA splicing remain largely unknown. Just as ribosome-binding antibiotics facilitated mechanistic studies of the ribosome, study of the catalytic mechanisms of the spliceosome could be enhanced by the availability of small molecule inhibitors that block spliceosome assembly and splicing at defined stages. We sought to identify inhibitors of Saccharomyces cerevisiae splicing by screening for small molecules that block yeast splicing in vitro. We identified 10 small molecule inhibitors of yeast splicing, including four antibiotics, one kinase inhibitor, and five oxaspiro compounds. We also report that a subset of the oxaspiro derivatives permitted assembly of spliceosomal complexes onto pre-mRNA but blocked splicing prior to the first cleavage reaction.