<p>Testicular steroidogenesis is fundamental to male reproductive health, but its disruption by environmental and emerging chemicals remains insufficiently characterized due to limitations in existing test systems. Traditional animal models pose ethical and logistical challenges, while the validated H295R assay—based on a female adrenal carcinoma cell line—fails to reflect male gonadal steroidogenesis. This review uses a semi-systematic approach to evaluate over 1500 studies employing in vitro models, including primary Leydig cells, Leydig cell lines, stem cell-derived Leydig-like cells, and advanced 3D testicular systems. We assess species origin, developmental relevance, culture conditions, and the extent to which these models replicate key steroidogenic pathways. Most models rely on rodent-derived, cancerous cell lines cultured in two-dimensional monolayers, with limited representation of human and immature Leydig cells. A targeted full-text analysis examined the effects of 23 reference chemicals on testosterone, progesterone, androstenedione, and estrogen levels across the H295R assay and eight testicular in vitro models. Forskolin, genistein, prochloraz, and ketoconazole showed consistent effects and may serve as promising reference compounds. However, data for most chemicals in testicular models are scarce or inconsistent—particularly for androstenedione and progesterone—underscoring the need for improved model standardization. We propose future directions to enhance predictive power, including the development of hormone-responsive, species- and stage-specific models cultured under hormone-controlled conditions. Such advances are essential to improve chemical safety assessment and facilitate regulatory acceptance of alternative test methods.</p>