Leucine plays crucial roles in protein proteolytic autophagy process, but the underlying mechanisms are not fully understood. Here, we combined in vitro reconstitution with comparative proteomics technology to identify the potential regulators for leucine-mediated autophagy pathway. We established a in vitro cell-free systems to mimic the activation of autophagy by monitoring the translocation of readout protein ATG14 to the autophagosome membrane. Based on the system, we further screened the potential regulators involved in the leucine-mediated autophagy pathway using comparative proteomics strategy.