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Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by the uncontrolled proliferation of aberrant myeloid blasts within the bone marrow, resulting in disrupted hematopoiesis and severe clinical consequences. Drug resistance represents a major barrier in AML treatment, frequently manifesting as relapse following initial remission with conventional chemotherapeutic agents such as cytarabine and venetoclax. The underlying mechanisms of drug resistance include enhanced drug efflux, altered drug metabolism, and activation of pro-survival signaling pathways, necessitating the elucidation of specific genetic determinants to enable the development of effective therapeutic strategies. The advent of CRISPR/Cas9 system has facilitated precise genomic modifications, permitting the generation of cell libraries with targeted gene knockouts in AML cells. This approach can identify genes whose disruption alters drug sensitivity, implicating their involvement in survival and resistance to cell death. This protocol outlines a systematic strategy to uncover genes associated with drug resistance in AML cells by leveraging CRISPR/Cas9-mediated functional genomic screening. By employing this methodology, genes conferring drug susceptibility upon knockout are noted as potential drivers of drug resistance, offering valuable insights for the rational design of targeted therapies.