The eukaryotic RNA exosome is a conserved and ubiquitous multiprotein complex that possesses multiple RNase activities and is involved in a diverse array of RNA degradation and processing events. While much of our current understanding of RNA exosome function has been elucidated using genetics and cell biology based studies of protein functions, in particular in S. cerevisiae, many important contributions in the field have been enabled through use of in vitro reconstituted complexes. Here, we present an overview of our approach to purify exosome components from recombinant sources and reconstitute them into functional complexes. Three chapters following this overview provide detailed protocols for reconstituting exosome complexes from S. cerevisiae, S. pombe, and H. sapiens. We additionally provide insight on some of the drawbacks of these methods and highlight several important discoveries that have been achieved using reconstituted complexes.