Antibody-drug conjugates (ADCs) targeting HER2 and TROP2 are now a mainstay of breast cancer therapeutics. ADCs equipped with cleavable linker-payload systems can kill non-target-expressing cancer cells through a bystander effect, which is influenced by antibody internalization and lysosomal degradation of the linker-payload complex. We first evaluated the IC 50 values of the HER2-targeting ADC trastuzumab deruxtecan (T-DXd) across a panel of breast cancer cell lines. HER2 protein expression levels correlated well with sensitivity to T-DXd, confirming that HER2 target expression is a major determinant of individual response. We next assessed the presence of bystander effect by co-culturing of MCF7 (low-HER2) with SK-BR-3 (HER2+) cells in culture. T-DXd successfully induced caspase-3 activation in MCF7. Leveraging observation in the co-culture experiment, we established a quantitative co-culture model to measure the magnitude of ADC-mediated bystander activity. In this model, we engineered the TNBC cell line MDA-MB-231 (HER2-null) to stably express EGFP to be co-cultured with SK-BR3 (HER2+). T-DXd was labeled with pHrodo Red to visualize lysosome-ADC contact in breast cancer cells, and DNA damage was assessed by gamma-H2AX staining. We observed internalization and co-localization to lysosome of T-DXd in selectively in SK-BR-3 cells, but not in MDA-MB-231 cells. Whereas, gamma-H2AX activation were detected in EGFP-tagged MDA-MB-231 cells, suggesting propagation of payload DXd via bystander effect. Finally, we performed genomic analyses to identify genetic factors potentially associated with bystander activity in 50 breast cancer patients treated with ADCs using targeted-panel NGS data. Alterations in NOTCH1, NOTCH2, and NF1 were associated with poor progression-free survival under ADC treatment by Cox regression analysis, and these genes are implicated in lysosomal and autophagy regulation. Collectively, these findings highlight that the anticancer efficacy of ADCs is substantially influenced by the bystander effect and underscore the need for deeper investigation into how genetic factors modulate this process.