Poly(A) tails are found at the 3′ end of almost every eukaryotic mRNA and are important for the stability of mRNAs and their translation into proteins. Thus, removal of the poly(A) tail, a process called deadenylation, is critical for regulation of gene expression. Most deadenylation enzymes are components of large multi-protein complexes. Here, we describe an in vitro deadenylation assay developed to study the exonucleolytic activities of the multi-protein Ccr4-Not and Pan2-Pan3 complexes. We discuss how this assay can be used with short synthetic RNAs, as well as longer RNA substrates generated using in vitro transcription. Importantly, quantitation of the reactions allows detailed analyses of deadenylation in the presence and absence of accessory factors, leading to new insights into targeted mRNA decay.