Eukaryotic deubiquitinase enzymes (DUBs) play a vital role in maintaining cellular homeostasis by deubiquitinating substrates marked for degradation. Of the ~100 DUBs discovered in humans, USP8 is unique for its association with human tumors including Cushing's disease (activating mutation), and solid cancers (genomic overexpression). The mechanisms underlying USP8 in tumorigenesis remain unknown. We first found that USP8 is essential for cell survival using CRISPR-Cas9-mediated homozygous excision and RNA interference. To circumvent obligate lethality, identify the substrates of USP8, and determine pathways dysregulated in human tumors with elevated USP8 activity, we engineered a GFP-auxin inducible degron (AID) at the endogenous USP8 locus in DLD1 cells. Additionally, we assayed both transient and long-term interactors using a promiscuous biotinylator BirA fused to a USP8 lentiviral construct. We performed cell survival, apoptosis assays, immunocytochemistry, western blot, RNAseq, and TMT labeled LC/MS, TUBE and IP-LC/MS study. We found that with auxin activation, DLD1USP8-GFP-AID had >80% reduction in USP8 within 30 minutes. At 6 hours, LC/MS revealed a profound alteration of the proteome and phosphoproteome with suppression of eukaryotic elongational initiation factor-2 (EIF2) signaling. We found that USP8 triggers the Integrated Stress Response (ISR), as measured by an ATF4 reporter assay. Consistently, USP8 overexpression led to a concordant elevation of EIF2 signaling and enrichment of EIF2 signaling peptides in cells expressing the USP8-BirA construct. Moreover, USP8 overexpression in PERK and GCN2 knockout lines suggests that USP8 activates ISR via both the amino acid deprivation (GCN2) and unfolded protein response (PERK) pathways. Taken together, our findings indicate that USP8 activation induces the Integrated Stress Response (ISR) through EIF2alpha phosphorylation and subsequent activation of the transcription factor ATF4. We propose that tumor cells hijack ISR signaling to promote pro-survival pathways, an effect that can be reversed by pharmacological inhibition of ISR using ISRIB. ISR pathway inhibition or decrease of USP8 activity shows a significant reduction of tumor volume in a syngeneic mouse model. These results reveal targetable downstream pathways of USP8 activation, offering potential therapeutic strategies to improve outcomes in patients with Chusing's disease and solid tumors.