In eukaryotes, pre-messenger RNA (pre-mRNA) cleavage and polyadenylation is one of the necessary processing steps that produce a mature and functional mRNA. Regulation on pre-mRNA cleavage and polyadenylation affects other processes such as mRNA translocation, stability, and translation. The process of pre-mRNA cleavage and polyadenylation, and its relationship with RNA splicing and translation, have been extensively studied due to its importance in vivo. A successful in vitro system has provided enormous amount of information to the study of cleavage and polyadenylation in the mammalian and yeast systems. Here, we describe an in vitro pre-mRNA cleavage system that faithfully cleaves pre-mRNA substrate using Arabidopsis cell/tissue cultures.