This study aimed to functionally validate genes associated with anthracycline-induced cardiotoxicity (AIC) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Through a comprehensive literature search, 80 genes significantly linked to AIC were identified, along with three additional genes (GSTM1, CBRI, and ERBB2). Among these, 38 genes were found to be expressed in human fetal heart, adult heart, and hiPSC-CMs. Using CRISPR/Cas9-based genome editing, each of these 38 genes was systematically knocked out, and the resulting doxorubicin-induced cardiotoxicity (DIC) phenotype was assessed. The results showed that knocking out 26 genes increased the susceptibility of hiPSC-CMs to DIC, while knocking out ATP2B1, HNMT, POR, CYBA, WDR4, and COL1A2 had no significant effect. Conversely, knocking out uptake transporters (SLC28A3, SLC22A17, and SLC28A1) demonstrated a protective effect against DIC. The study provides a comprehensive platform for functional validation of DIC-associated genes, offering insights into their potential mechanistic roles and paving the way for future studies on variant associations and cardioprotective drug development.This study aimed to functionally validate genes associated with anthracycline-induced cardiotoxicity (AIC) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Through a comprehensive literature search, 80 genes significantly linked to AIC were identified, along with three additional genes (GSTM1, CBRI, and ERBB2). Among these, 38 genes were found to be expressed in human fetal heart, adult heart, and hiPSC-CMs. Using CRISPR/Cas9-based genome editing, each of these 38 genes was systematically knocked out, and the resulting doxorubicin-induced cardiotoxicity (DIC) phenotype was assessed. The results showed that knocking out 26 genes increased the susceptibility of hiPSC-CMs to DIC, while knocking out ATP2B1, HNMT, POR, CYBA, WDR4, and COL1A2 had no significant effect. Conversely, knocking out uptake transporters (SLC28A3, SLC22A17, and SLC28A1) demonstrated a protective effect against DIC. The study provides a comprehensive platform for functional validation of DIC-associated genes, offering insights into their potential mechanistic roles and paving the way for future studies on variant associations and cardioprotective drug development.