Mitochondria, the powerhouses of the cell, are central to tumor initiation and growth, yet their protein interactions in living tumors remain poorly understood. Although crosslinking mass spectrometry (XL-MS) has provided valuable insights in cultured cells and isolated tissues, in vivo application is limited by poor crosslinker delivery. Here, we present an approach that uses intratumoral injection of crosslinker-loaded nanoparticles in xenografted mice to enable in vivo DSS crosslinking of tumor mitochondria, thereby facilitating XL-MS–based analysis of mitochondrial protein complexes. We identified 2,368 crosslinked peptides from 654 mitochondrial proteins, covering 58% of the human mitochondrial proteome. Among these, 553 crosslinked peptides were mapped to proteins involved in thermogenesis, ribosome function, and branched-chain amino acid degradation—metabolic pathways closely linked to tumor progression. Structural concordance was high (91%) with PDB and AlphaFold models, and complementary conformational insights were obtained, including previously missing data on the DAPIT subunit of ATP synthase, thus complementing cryo-electron microscopy (cryo-EM) structures. We further mapped 217 mitochondrial PPIs, including 146 not annotated in STRING, highlighting the influence of the tumor microenvironment on protein interactions in vivo and providing valuable insights into tumor biology.