Macroautophagy (hereafter referred to as autophagy) is a degradation system that delivers cytoplasmic materials to lysosomes via autophagosomes. Autophagic flux is defined as a measure of autophagic degradation activity. Despite several methods for monitoring autophagic flux being currently utilized, interest in finding a highly accurate, sensitive and well-quantifiable assay is still growing. Therefore, we introduce a new approach analyzing autophagic flux in vitro and in vivo using enzyme-linked immunosorbent assay (ELISA) technique. In order to adapt this assay from LC3-II turnover measured by Western blot in the presence and absence of lysosomal inhibitors, we induced autophagy by starvation or rapamycin and mitophagy (mitochondrial degradation by autophagy) by CCCP in C2C12 myotubes for 8 h and in mice for 48 h with and without Bafilomycin A1 or colchicine treatment, respectively. Following subcellular fractionation of mouse skeletal muscle cells and tissue, cytosolic, membrane, and mitochondrial fractions were analyzed through a sandwich ELISA using two LC3 antibodies, LC3 capture and HRP-conjugated LC3 detection antibodies. Using this ELISA, changes in the membrane-bound or mitochondrion-associated LC3-II levels, and the ratio of the LC3-II from each fraction to LC3-I levels (cytosolic fraction) were evaluated for measuring autophagy and mitophagy flux. This study demonstrates that this ELISA was more sensitive and reliable to measure autophagic/mitophagic flux in both in vitro and in vivo, compared with the most commonly used LC3 turnover assay via Western blot. © 2017 Elsevier Inc.