RNA molecules contain many chemical modifications that can regulate a variety of biological processes. Messenger RNA (mRNA) molecules are critical components in the central dogma of molecular biology. The discovery of reversible chemical modifications in eukaryotic mRNA brings forward a new research field in RNA modification-mediated regulation of gene expression. The modifications in mRNA generally exist in low abundance. The use of highly pure mRNA is critical for the confident identification of new modifications as well as for the accurate quantification of existing modifications in mRNA. In addition, isolation of highly pure mRNA is the first step in many biological research studies. Therefore, the methods for isolating highly pure mRNA are important for mRNA-based downstream studies. A variety of methods for isolating mRNA have been developed in the past few decades and new methods continuously emerge. This review focuses on the methodologies and protocols for isolating mRNA populations. In addition, we discuss the advantages and limitations of these methods. We hope this paper will provide a general view of mRNA isolation strategies and facilitate studies that involve mRNA modifications and functions.