A high level of microsatellite instability (MSI-H+) is an emerging predictive and prognostic biomarker for immunotherapy response in cancer. Recently, MSI-H+ has been detected in a variety of cancer types, in addition to the classical cancers associated with Lynch Syndrome. Clinical testing for MSI-H+ is currently performed primarily through traditional polymerase chain reaction (PCR) or immunohistochemistry (IHC) assays. However, next-generation sequencing (NGS)–based approaches have been developed which have multiple advantages over traditional assays. For instance, NGS has the ability to interrogate thousands of microsatellite loci compared with just 5–7 loci that are detected by PCR. In this chapter, we detail the biochemical and computational steps to detect MSI-H+ from analysis of paired tumor and normal samples through NGS. We begin with DNA extraction, describe sequencing library preparation and quality control (QC), and outline the bioinformatics steps necessary for sequence alignment, preprocessing, and MSI-H+ detection using the software tool MANTIS. This workflow is intended to facilitate more widespread usage and adaptation of NGS-powered MSI detection, which can be eventually standardized for routine clinical testing.