Significance We use a protein labeling technique based on an engineered ascorbate peroxidase (APEX) to map the proteome of the mitochondrial matrix in live tissues. The approach allows us to establish MitoMax, a comprehensive database providing a high-quality inventory of Drosophila mitochondrial proteins with subcompartmental annotation. We demonstrate that APEX labeling is effective in vivo and provides an opportunity to characterize subcellular proteomes in specific cell types and in different physiological conditions. Given the interest in defining the mitochondrial proteome in different physiological conditions and tissues, our analysis provides a resource for systematic functional analyses of mitochondria that will in particular facilitate investigation of mitochondrial diseases.