LAUSR

&NA; N6‐methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). Until now, two RNA demethylases have been identified, including FTO (fat mass and obesity‐associated protein) and ALKBH5 (&agr;‐ketoglutarate‐dependent dioxygenase alkB homologue 5). As a mammalian m6A demethylase, ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles, and ALKBH5 may play a significant role in these biological processes. Nevertheless, no modulator of ALKBH5 has been reported. The reason for that may be the lack of in vitro assays for ALKBH5 inhibitor screening. Herein, we describe the development of two homogeneous assays for ALKBH5 using N6‐methyladenosine as substrate with different principles. Using ALKBH5 recombinant, we developed a formaldehyde dehydrogenase coupled fluorescence based assay and an antibody based assay for the activity evaluation of ALKBH5. These robust coupled assays are suitable for screening ALKBH5 inhibitors in 384‐well format (Z' factors of 0.74), facilitating the discovery of modulators in the quest for the regulation of biological processes. HighlightsTwo methods with different principles were first developed for the activity quantification of ALKBH5.Two methods for ALKBH5 activity were optimized and kinetic parameters were determined.Both assays could reliably serve as valuable and efficient HTS tools to discover and evaluate ALKBH5 inhibitors.