Endocrine pathways are crucial in regulating physiological functions in organisms, including growth and development, metabolism, tissue function, and reproduction. Exposure to endocrine disrupting chemicals (EDCs) can interfere with normal hormonal function and lead to adverse effects in organisms and/or their offspring. As a result, regulatory testing and assessment requirements have been implemented to identify and regulate EDCs-defined by the World Health Organization as chemicals that alter the function of an endocrine system and cause "subsequent adverse effects in an intact organism, its progeny, or (sub)populations" - although the approaches taken for assessment vary worldwide. As such, the current typical ecotoxicological assessment paradigm for EDCs involves initial testing for endocrine activity using in vitro and lower-tier in vivo assays, followed by higher-tier in vivo testing which can provide additional endocrine mechanistic data and establishes any consequent adversity. The Organisation for Economic Cooperation and Development and the United States Environmental Protection Agency, among other entities, have validated and adopted standardized in vivo test guidelines for mammals, fish, and amphibians. These tests can be challenging and take months to perform, and many require the use of large numbers of laboratory animals. This paper summarizes the current state of the science for evaluating the endocrine disrupting potential of chemicals in fish and amphibians using in vivo test guideline assays across the estrogen, androgen, thyroid, and steroidogenesis modalities. Current challenges associated with conducting and interpreting the in vivo assays are discussed. Opportunities and potential next steps to strengthen this growing area of testing are explored, including potential to inform development and application of new approach methodologies and thereby reduce reliance on using laboratory animals.