In eukaryotes, transcription of genomic DNA produces precursors to messenger RNAs (pre-mRNAs) which contain both expressed regions (exons) and intervening non-coding regions (introns). Pre-mRNA splicing, the process of exon ligation with concurrent intron excision via two transesterification reactions, is carried out by the spliceosome: a highly dynamic macromolecular complex consisting of five core small nuclear ribonucleoprotein particles (snRNPs) and many transiently associated proteins. While much insight into the mechanism of splicing has come from in vivo experiments and ensemble in vitro assays, these studies are generally limited in their focus to stable, readily identifiable spliceosomal complexes. To overcome this barrier, this thesis reports on experiments using single molecule fluorescence resonance energy transfer (FRET) and colocalization single molecule spectroscopy (CoSMoS) methods to detect pre-mRNA structural rearrangements during the splicing reaction and to investigate their timing with respect to the steps of spliceosome assembly in budding yeast (Saccharomyces cerevisiae). To do this, a pre-mRNA containing two site-specifically incorporated fluorophores was prepared. The FRET acceptor (Cy5) was placed within the 5' exon in proximity to the 5' splice site, whereas the FRET donor (Cy3) was located just upstream of the intron branch point nucleotide that provides the nucleophile for the first transesterification reaction. By monitoring changes in FRET efficiency on single pre-mRNAs during splicing reactions containing this substrate, conformational changes within the pre-mRNA were observed coincident with the assembly of the spliceosome. Further, by stalling the spliceosome at defined points during the splicing reaction, discrete FRET efficiencies were correlated with association of particular snRNPs. The data indicate that in this yeast splicing system, there is substantial separation (100 Å or greater) of the 5' splice site from the branch site through the second stable spliceosomal assembly complex: the A-Complex spliceosome. Collectively these experiments have provided insight into the stages of spliceosome assembly and activation during which the branch site and 5' splice site come into spatial proximity, a necessary precursor to catalysis.