Based on our research on the regulation of mitochondrial quality control and the functional mechanisms of the deubiquitinase USP30; here, we utilized HeLa cells stably expressing USP30-Flag to systematically identify its interacting proteome through affinity purification-mass spectrometry (AP-MS). This screening successfully enriched a series of high-confidence proteins that potentially interact with USP30 within the mitochondrial environment. The experimental verification of candidate proteins through co-immunoprecipitation highlighted the robust capability of this AP-MS strategy in capturing functional interactors, providing a molecular basis for further understanding USP30-mediated regulation of mitochondrial proteostasis.