Ubiquitin-specific protease 30 (USP30) is a deubiquitylating enzyme (DUB) localized in the mitochondrial membrane, which is related to PINK1/Parkin-mediated mitophagy, pexophagy, BAX/BAK-dependent apoptosis, and IKKβ-USP30-ACLY-regulated lipogenesis/tumorigenesis. Mission therapeutics pointed their in-house screened MTX652 as USP30 inhibitor for Phase I clinical trial in early 2022. Activity-based probes (ABPs) provide a powerful tool for screen USP30 inhibitors. Here, we report the first small molecule ABPs (ABP 2 and ABP 4) for profiling activity of USP30. Through in-gel fluorescence, target-enrichment and proteomics analysis, we demonstrate that ABP 2 and ABP 4 selectively engage USP30 at nanomolar concentration for only 10 min incubation time in live cells. This cellular USP30-engagement is selectively depending on the catalytic cysteine of USP30. Interestingly, DESI1 and DESI2, the small ubiquitin-related modifier (SUMO) proteases, are also engaged by ABP 2 and ABP 4, providing the novel strategy for these probes as DESIs ABPs. We use proteomics analysis to identify the probes targeted proteins by DIA analysis.