Supplementation of branched chain amino acids, especially leucine, is critical to improve malnutrition by regulating protein synthesis and degradation. Emerging evidence has linked leucine deprivation induced protein breakdown to autophagy. In this study, we aimed to establish a cell-free assay recapitulating leucine-mediated autophagy in vitro and dissect its biochemical requirement. We found that in a cell-free assay, membrane association of Barkor/Atg14(L), a specific autophagosome-binding protein, is suppressed by cytosol from nutrient-rich medium and such suppression is released by nutrient deprivation. We also showed that rapamycin could efficiently reverse the suppression of nutrient rich cytosol, suggesting an essential role of mTORC1 in autophagy inhibition in this cell-free system. Furthermore, we demonstrated that leucine supplementation in the cultured cells blocks Barkor puncta formation and autophagy activity. Hence, we establish a novel cell-free assay recapitulating leucine-mediated autophagy inhibition in an mTORC1-dependent manner; this assay will help us to dissect the regulation of amino acids in autophagy and related human metabolic diseases.