The yeast Saccharomyces cerevisiae provides an ideal model system for elucidation of the molecular mechanisms that regulate the nonsense-mediated mRNA decay (NMD) pathway. This chapter describes an array of molecular biological, genetic, and biochemical tools that facilitate the characterization of transcripts that comprise NMD substrates and provide insights into the roles of the upf/nmd proteins in mRNA decay and translation termination. Examples illustrate the use of these methods in wild-type and NMD-deficient cells to monitor the abundance, structure, and half-lives of nonsense-containing mRNAs, the read through of premature termination codons by the ribosome, and the positioning of ribosomes at or near normal and premature termination codons.