Cells of the monocytic lineage are involved in the pathophysiology of several human cancers. In myeloid hematologic malignancies such as chronic myelomonocytic leukemia (CMML) and monocytic AML (M4/M5 FAB subtypes), leukemic monocytes display poor response to standard of care treatments and dismal complete remission rates. In solid tumors, tumor-associated macrophages (TAMs) are critical cells of monocytic origin that promote an immunosuppressive tumor microenvironment in malignancies such as TNBC, PDAC, and GBM, thereby driving tumor growth and resistance to PD-1/PD-L1 blockade. Interestingly, both leukemic monocytes and TAMs share a highly differentiated phenotype and a low proliferative capacity, which render them refractory to the mechanism of action (MOA) of established chemotherapeutic regimens. Therefore, identifying druggable targets with broad expression across the monocytic maturation lineage is a promising strategy for designing antibody-drug conjugates (ADCs) aimed at overcoming monocyte-driven resistance. In this work, we leveraged OncoPrecision's Patient Micro-Avatar (PMA) ex vivo platform to validate novel targets and the optimal payload to develop an ADC to target leukemic monocytes and TAMs. This screening campaign identified CD64 as a target with high expression and druggability in patient-derived monocytic-cells and unveiled PNU-159682 (PNU) as the payload with the optimal MOA to deplete monocytic cells. Intriguingly, payloads with proven clinical success in other malignancies, such as Exatecan and MMAE, displayed poor/null activity against leukemic monocytes and TAMs, thus highlighting a non-obvious vulnerability of monocytic cells to PNU. ONC001, our first-in-class ADC conjugated to PNU through a non-cleavable linker displays picomolar activity against a panel of monocytic cell lines in vitro and against patient-derived leukemic monocytes and TAMs ex vivo. Multiple in vivo models of leukemia and solid tumors also confirmed a remarkable efficacy and selectivity of ONC001, including a humanized mouse model in which TAMs from the TME of established xenografted tumors were efficiently depleted after a single injection of the ADC. Importantly, we extensively investigated the safety of ONC001 by evaluating its on-target and off-target activity in multiple animal models, and observed remarkable tolerability with no signs of toxicity. Together, these preclinical findings demonstrate a favorable therapeutic index for ONC001, supporting its potential as a powerful agent for depleting pathogenic monocytes in cancer treatment.