Proximity labeling is an in situ protein biotinylation reaction by enzymatically generated reactive biotin-species which have a short lifetime (less than 1 ms) in aqueous solution. Recently, proximity labeling has shown remarkable new findings in biological research fields such as sub-organelle proteome mapping and in vivo interactome identification which have not been easily obtained by conventional methods. Our group recently developed an improved proximity labeling method (Spot-ID) by mass-spec detection of the biotin-labeled proteins which are generated by engineered ascorbate peroxidase (APEX) or promiscuous biotin ligase (pBirA). This method allows the direct identification of the labeled protein without false positive findings and the structural identification of the labeled protein in live cells. Using this method, we could map in vivo proteomic architecture of the inner mitochondrial membrane (IMM) whose membrane topology has not been fully characterized yet. Furthermore, this method allowed the identification of new elements in the rapamycin-induced interactome on the FK506-rapamycin binding (FRB) domain of mTOR in living cells. In this seminar, I will introduce a new approach to perform proximity crosslinking by utilization of a simple coupling reaction in live cells. This method could identify physically interacting molecular partner(s) of protein of interest in live cells. Overall, we could observe that these new methods are very useful to unveil systems biology in live cells.