In vitro assays have provided a valuable tool to study the mechanism of 3' processing of eukaryotic mRNA precursors and have contributed a great deal to the identification of factors that carry out and regulate 3' processing. Previously， we have shown that transcriptional activators directly enhance polyadenylation by utilizing in vitro transcription-coupled polyadenylation with the prototypical transcription activator GAL4-VP16. In this chapter， we describe a detailed protocol for this assay， which will be useful in examining potential roles for other transcription-related factors in 3' processing and other questions related to the coupling of transcription and mRNA polyadenylation.