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Aptamers are small, functional nucleic acids that bind a variety of targets, often with high specificity and affinity. Genomic aptamers constitute the ligand-binding domains of riboswitches, whereas synthetic aptamers find applications as diagnostic and therapeutic tools, and as ligand-binding domains of regulatory RNAs in synthetic biology. Discovery and characterization of aptamers has been limited by a lack of high-throughput approaches that uncover the target-binding domains and the biochemical properties of individual sequences. With the advent of high-throughput sequencing, large-scale analysis of in vitro selected populations of aptamers (and catalytic nucleic acids, such as ribozymes and DNAzmes) became possible. In recent years the development of new experimental approaches and software tools has led to significant streamlining of the selection-pool analysis. This article provides an overview of post-selection data analysis and describes high-throughput methods that facilitate rapid discovery and biochemical characterization of aptamers.