This article contains a protocol for selective 2'-hydroxyl acylation analyzed by primer extension and sequencing (SHAPE-Seq) that, through a combination of structure-dependent chemical probing and next-generation sequencing technologies, achieves structural characterization of hundreds of RNAs in a single experiment. This protocol is applicable in a variety of conditions, and represents an important tool for understanding RNA biology. The protocol includes methods for the design and synthesis of RNA mixtures for study, and the construction and analysis of structure-dependent sequencing libraries that reveal structural information of the RNAs in the mixtures. The methods are generally applicable to studying RNA structure and interactions in vitro in a variety of conditions, and allows for the rapid characterization of RNA structures in a high-throughput manner.