Current methods for intracellular protein analysis mostly require the separation of specific organelles or the change of intracellular environment. However, the functions of these proteins are determined by their native microenvironment as they usually form complexes with ions, nucleic acids, and other proteins. Thus, we have explored a method for in situ crosslinking and mapping of mitochondrial proteins in living cells. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are functionalized with dihexadecyldimethylammonium bromide (DDAB) to deliver protein crosslinkers into mitochondria. In situ crosslinked proteins are analyzed using mass spectrometry.