<p>Liver steatosis resulting from chemical exposure is a growing clinical concern, pointing to the need for early detection in chemical safety assessments. New approach methodologies (NAMs) based on adverse outcome pathway (AOPs) networks are emerging tools to assess the safety of chemicals as an alternative to animal studies. Amongst others, NAM can include a human-based in vitro system linked to a battery of complementary assays, each measuring an individual key event within the respective AOP network. These AOP-based NAMs allow to acquire mechanistic insight, while facilitating the translation of in vitro data into outcomes relevant to hazard assessment. The current review provides pragmatic guidance for developing NAMs entailing an AOP network-driven in vitro testing platform suitable for steatogenic hazard assessment of chemicals, intended for use in academic, industrial and regulatory settings. In a first part, underlying mechanisms for highly specific key events for liver steatosis are described. In a second part, a workflow is provided for the establishment of NAMs, including the selection of human-based in vitro cell models, functional in vitro assays, reference control chemicals, exposure and treatment conditions. In a third part, future perspectives for AOP-based liver steatosis risk assessment are outlined.</p>