Internal ribosome entry sites (IRESs) substitute RNA sequences for some or all of the canonical translation initiation protein factors. Therefore, an important component of understanding IRES function is a description of the three-dimensional structure of the IRES RNA underlying this mechanism. This includes determining the degree to which the RNA folds, the global RNA architecture, and higher resolution information when warranted. Knowledge of the RNA structural features guides ongoing mechanistic and functional studies. In this chapter, we present a roadmap to structurally characterize a folded RNA, beginning from initial studies to define the overall architecture and leading to high-resolution structural studies. The experimental strategy presented here is not unique to IRES RNAs but is adaptable to virtually any RNA of interest, although characterization of RNA-protein interactions requires additional methods. Because IRES RNAs have a specific function, we present specific ways in which the data are interpreted to gain insight into that function. We provide protocols for key experiments that are particularly useful for studying IRES RNA structure and that provide a framework onto which additional approaches are integrated. The protocols we present are solution hydroxyl radical probing, RNase T1 probing, native gel electrophoresis, sedimentation velocity analytical ultracentrifugation, and strategies to engineer RNA for crystallization and to obtain initial crystals.