RNA helicases have fascinated and puzzled investigators for the three decades that have passed since the first report on an RNA helicase, as a "protein involved in ATP-dependent binding of mRNA." Since then, it has become clear that RNA helicases are ubiquitous, highly conserved enzymes that participate in virtually all aspects of RNA metabolism from bacteria to human. In eukaryotic RNA metabolism, RNA helicases are the largest class of enzymes, and many of these proteins have emerged as central players in the regulation of gene expression at the RNA level. This volume of Methods in Enzymology aims to provide a reference for the diverse, powerful tools used to analyze RNA helicases. The contributions in this volume cover the broad scope of methods in the research on these enzymes. Several chapters describe quantitative biophysical and biochemical approaches to study molecular mechanisms and conformational changes of RNA helicases. Further chapters cover structural analysis, examination of co-factor effects on several representative examples, and the analysis of cellular functions of select enzymes. Two chapters outline approaches to the analysis of inhibitors that target RNA helicases.