Most mitochondrial proteins are translated in the cytosol and imported into mitochondria. Mutations in the mitochondrial protein import machinery cause human pathologies. However, a lack of suitable tools to measure protein uptake across the mitochondrial proteome has prevented the identification of specific proteins affected by import perturbation. Here, we introduce mePRODmt, a pulsed-SILAC based proteomics approach that includes a booster signal to increase the sensitivity for mitochondrial proteins selectively, enabling global dynamic analysis of endogenous mitochondrial protein uptake in cells. We applied mePRODmt to determine protein uptake kinetics and examined how inhibitors of mitochondrial import machineries affect protein uptake. Monitoring changes in translation and uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated by the import and translation machineries via activation of the integrated stress response. Strikingly, uptake changes were not uniform, with subsets of proteins being unaffected or decreased due to changes in translation or import capacity. [Display omitted] • Proteomics approach to quantify protein uptake into mitochondria • Determination of protein uptake rates for >700 mitochondrial proteins • Characterization of differential protein uptake changes during mitochondrial stress • Protein translation and translocation integrate to change protein uptake Schäfer et al. describe a proteomics method to quantify mitochondrial protein uptake for >700 mitochondrial proteins. They characterize how protein import inhibitors affect protein uptake. This revealed that changes in protein translation and translocation integrate to control mitochondrial uptake for different sets of proteins and submitochondrial compartments. [ABSTRACT FROM AUTHOR]