Pyrethroids, which are widely utilized in agriculture, household products, and public health for their potent insecticidal properties, elicit significant concerns regarding their potential endocrine-disrupting effects. However, previous studies have yielded inconsistent data, largely due to the absence of a standardized screening system. To address this limitation, the present study introduces an Integrated Approach to Testing and Assessment (IATA) to evaluate the endocrine-disrupting potential of pyrethroids, aligned with the Adverse Outcome Pathway (AOP) framework. Employing this IATA-based methodology, the endocrine-disrupting effects of five pyrethroids, allethrin, phenothrin, deltamethrin, cypermethrin, and lambda-cyhalothrin-were investigated, with a focus on hormone levels of 17β-estradiol (E2) and testosterone (T). Enzyme-linked immunosorbent assays (ELISA) and receptor transactivation assays were utilized to assess the direct receptor interactions and alternative disruption mechanisms. The results demonstrated that lambda-cyhalothrin and phenothrin significantly elevated E2 levels, while all tested compounds substantially reduced T levels. Notably, transactivation assays indicated that these pyrethroids function as estrogenic agonists and androgenic antagonists, suggesting their complex role in endocrine disruption. The IATA-based framework, incorporating steroidogenesis and receptor transactivation assays, provides a comprehensive approach for assessing endocrine disruption, enabling the early identification and prioritization of hazardous chemicals. By predicting adverse outcomes without relying on in vivo testing, this integrated approach enhances regulatory decision making, promotes public health protection, and supports ethical and efficient chemical risk assessment.