Ubiquitin-specific protease 22 (USP22) regulates epigenetic gene expression by deubiquitinating histone H2B (H2Bub1) and upregulating oncogenic proteins and pathways, while antagonizing p53-mediated tumor suppression. USP22 is frequently overexpressed in cancers and associated with therapy resistance and poor prognosis yet remains largely untargeted pharmacologically. Here, using a fluorescence-based USP22 deubiquitinase assay to screen the LOPAC^®1280 library, we identified β-Lapachone, a natural ortho-naphthoquinone with strong anticancer activities, as a potent USP22 inhibitor. β-Lapachone potently inhibited USP22 enzymatic activity, with a half-maximal inhibitory concentration (IC₅₀) of ~0.75 μM, and molecular docking revealed its occupation of the catalytic pocket adjacent to the USP22 active-site triad, supporting a potential binding mode. Functionally, β-Lapachone suppressed proliferation and induced apoptosis in A549 and H1299 RAS-mutant lung adenocarcinoma (LUAD) cells, while USP22 knockout conferred marked resistance, indicating partial USP22 dependence. In patient-derived LUAD models, β-Lapachone inhibited sphere formation and reduced CD133⁺ cancer stem cell populations. Notably, it synergized with cisplatin to enhance DNA damage and apoptosis. In vivo, β-Lapachone significantly suppressed tumor growth in a syngeneic KRAS-mutant/p53-Null mouse lung cancer model and further potentiated cisplatin-induced antitumor effects. Collectively, these findings identify β-Lapachone as a potent inhibitor of USP22 and validate USP22 inhibition as a key mechanism underlying its anticancer activity in LUAD cells, both in vitro and in vivo.