Organelle communication is largely mediated by proteins at membrane contact sites (MCS), such as the endoplasmic reticulum (ER)-mitochondria, ER-plasma membrane (PM), and mitochondria-lipid droplets (LD). In this project, we developed a new tool for identifying membrane proteins at MCS by utilizing two mutually orthogonal binding pair systems, streptavidin-biotin (SA-Bt) and cucurbit[7]uril-adamantane (CB[7]-Ad), in conjunction with two distinct engineered enzymes, TurboID and APEX2. This enabled us to successfully identify proteins at the contact sites of the ER and mitochondria, and further allowed us to detect protein sets that undergo structural and locational changes at the ER-mitochondria contact site during the process of mitophagy.