Summary Cellular mRNAs are susceptible to a variety of chemical modifications that play important roles in regulating gene expression and cellular function. The most abundant internal mRNA modification is m6A, which occurs when adenosine residues become methylated. m6A influences nearly every aspect of the mRNA life cycle and is critical for a variety of physiological processes, such as gametogenesis, neurodevelopment, learning and memory, immune regulation, and stem cell proliferation and differentiation. Additionally, m6A has emerged as an important RNA regulatory mechanism during cancer progression: altered levels of m6A readers, writers, and erasers are observed in several human cancers, and methylation of oncogenes and tumor suppressors has been shown to impact their expression and promote cancer development. Thus, there is a great need both for understanding how m6A is regulated in cells as well as for developing drugs that target the m6A methyltransferase machinery. However, a major limitation has been the lack of simple, cost-effective methods for detecting changes in m6A methylation in cells in a manner that is compatible with high-throughput screening (HTS). Here, we overcome this barrier by developing a novel genetically encoded m6A sensor which provides a simple fluorescent readout for m6A methylation in living cells. Aim 1 will optimize the m6A sensor system and generate cellular tools to facilitate using the system for diverse applications. Aim 2 will develop the m6A sensor into a HTS-compatible system and demonstrate its utility for drug discovery efforts by performing a pilot HTS designed to identify m6A methyltransferase inhibitors. Aim 3 will create molecular tools incorporating the system and measure the performance of the m6A sensor in vivo. Altogether, these studies will provide a much-needed tool for detecting m6A dynamics in cells and will develop an optimized system for HTS-based studies of m6A methylation. Our technology is likely to have an immediate impact both for basic investigations of m6A biology in cancer as well as drug discovery efforts aimed at identifying novel m6A methyltransferase inhibitors.