Next-generation sequencing is a powerful method to interrogate the nucleotide composition for millions of DNA strands simultaneously. This technology can be utilized to profile microRNAs from multiple origins, such as tissues, cells, and body fluids. Next-generation sequencing is increasingly becoming a common and readily available technique for all laboratories. However, the bottleneck for next-generation sequencing is not within the laboratory but with the bioinformatics and data analysis of next-generation sequencing data. This chapter briefly describes the methods used to prepare samples for next-generation sequencing within the laboratory, before a deeper description of the methods used for data analysis.