mRNA decapping is a common step shared between two important mRNA decay pathways in yeast, Saccharomyces cerevisiae. To investigate how mRNAs are decapped, we have developed an assay that can be easily used to measure the decapping activity. This assay has been used to isolate yeast strains with altered decapping activities. The results demonstrated that decreased decapping activity in vitro corresponds well with the decapping-deficient phenotype in vivo. This assay has been applied to the purified yeast decapping enzyme Dcp1 protein as well as crude yeast extracts and Xenopus oocyte extracts.