Obtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it was insufficiently specific when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine newly discovered mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible. Display omitted •Ratiometric tagging with an engineered peroxidase gives nanometer spatial resolution•Mitochondrial intermembrane space proteome mapped with >94% specificity•Nine newly discovered mitochondrial proteins confirmed by imaging and western blotting•Data support MICU1 and MICU2 localization in the mitochondrial intermembrane space APEX is an engineered ascorbate peroxidase that can tag endogenous proteins for identification by mass spectrometry. Here, Hung et al. improve spatial specificity of APEX and analyze protein composition of the mitochondrial intermembrane space (IMS). The resulting IMS proteome of 127 proteins has >94% mitochondrial specificity and includes 9 newly discovered, validated mitochondrial proteins.