Pre-mRNA splicing is a key step for generating mature protein-coding mRNA. An RNA-protein complex known as the spliceosome carries out the chemistry of pre-mRNA splicing. However, several pre-spliceosomal intermediates are assembled on the pre-mRNA before the formation of the catalytically activated spliceosome. The progression to the activated spliceosome involves a cascade of the rearrangement events of the RNA-RNA, RNA-protein, and protein-protein interactions within the pre-spliceosomal intermediates. These rearrangements generate multiple combinatorial interactions of the spliceosome with the substrate, which enhances the accuracy of the splice site selection. Each rearrangement also represents a step at which splicing can potentially be subjected to regulation. The aim of this chapter is to provide an overview of the components of the spliceosome and their rearrangements along the spliceosome assembly pathway.