An essential aspect of protein quality control is enzymatic removal of membrane proteins from the lipid bilayer. Failures in this essential cellular process are associated with neurodegenerative diseases and cancer. Msp1 is a AAA+ ( A TPases A ssociated with diverse cellular A ctivities) protein that removes mistargeted proteins from the outer mitochondrial membrane (OMM). How Msp1 selectively recognizes and extracts substrates within the complex OMM ecosystem, and the role of the lipid bilayer on these processes is unknown. Here, we describe the development of fully defined, rapid, and quantitative extraction assay that retains physiological substrate selectivity. Using this new assay, we systematically modified both substrates and the lipid environment to demonstrate that Msp1 recognizes substrates by a hydrophobic mismatch between the substrate TMD and the lipid bilayer. We further demonstrate that the rate limiting step in Msp1 activity is extraction of the TMD from the lipid bilayer. Together, these results provide foundational insights into how the lipid bilayer influences AAA+ mediated membrane protein extraction.