The major cytoplasmic 5' to 3'-exoribonuclease activity is carried out by the Xrn1 protein in eukaryotic cells. A number of different approaches can be used to study multifunctional Xrn1 protein activity in vitro. In this chapter, we concentrate on methods used in our laboratory to analyze Xrn1 5' to 3'-exoribonuclease activity. Some of these techniques may also be suitable for detecting 3' to 5'-exoribonuclease or endoribonuclease activity. For these reasons, these assays can be used to isolate new proteins with ribonuclease activity and, when performed in combination with in vivo experiments, will contribute to a new level of understanding of the function of these factors.