Atg4 is required for cleaving Atg8, allowing it to be conjugated to 
phosphatidylethanolamine on phagophore membranes, a key step in autophagosome 
biogenesis. Deconjugation of Atg8 from autophagosomal membranes could be also a 
regulatory step in controlling autophagy. Therefore, the activity of Atg4 is 
important for autophagy and could be a target for therapeutic intervention. In 
this study, a sensitive and specific method to measure the activity of two Atg4 
homologs in mammalian cells, Atg4A and Atg4B, was developed using a fluorescence 
resonance energy transfer (FRET)-based approach. Thus LC3B and GATE-16, two 
substrates that could be differentially cleaved by Atg4A and Atg4B, were fused 
with CFP and YFP at the N- and C-terminus, respectively, allowing FRET to occur. 
The FRET signals decreased in proportion to the Atg4-mediated cleavage, which 
separated the two fluorescent proteins. This method is highly efficient for 
measuring the enzymatic activity and kinetics of Atg4A and Atg4B under in vitro 
conditions. Applications of the assay indicated that the activity of Atg4B was 
dependent on its catalytic cysteine and expression level, but showed little 
changes under several common autophagy conditions. In addition, the assays 
displayed excellent performance in high throughput format and are suitable for 
screening and analysis of potential modulators. In summary, the FRET-based assay 
is simple and easy to use, is sensitive and specific, and is suitable for both 
routine measurement of Atg4 activity and high-throughput screening.