AbstractRNA modifications and their corresponding epitranscriptomic writer and eraser enzymes regulate gene expression. Altered RNA modification levels, dysregulated writers, and sequence changes that disrupt epitranscriptomic marks have been linked to mitochondrial and neurological diseases, cancer, and multifactorial disorders. The detection of epitranscriptomics marks is challenging, but different next generation sequencing (NGS)‐based and mass spectrometry‐based approaches have been used to identify and quantitate the levels of individual and groups of RNA modifications. NGS and mass spectrometry‐based approaches have been coupled with chemical, antibody or enzymatic methodologies to identify modifications in most RNA species, mapped sequence contexts and demonstrated the dynamics of specific RNA modifications, as well as the collective epitranscriptome. While epitranscriptomic analysis is currently limited to basic research applications, specific approaches for the detection of individual RNA modifications and the epitranscriptome have potential biomarker applications in detecting human conditions and diseases.This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Processing > tRNA Processing RNA in Disease and Development > RNA in Disease