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Abstract Endocrine disruptors (EDs) are chemical substances that interfere with the endocrine system, adversely affecting human health and environment. Legislation with aim to eliminate and ban EDs have been introduced in EU, but the identification of EDs remains challenging and crucial step towards regulation and risk management. A guidance for ED assessment has been recently established for pesticides and biocides in the EU, which heavily relies on traditional toxicological testing in vivo. Most notably lacking mechanistic methods for some ED modalities and not covering many other modalities that might be affected by EDs. In this project, we focus on the ED assessment according to the valid legislation and explore the possibility to employ alternative methods to bolster the mechanistic understanding of the ED effects and eventually decrease the need for in vivo testing. We selected a well‐studied industrial chemical perfluorooctanesulfonic acid (PFOS) to be a model compound in a case study for ED assessment where the EU criteria were applied in the frame of human health risk assessment with focus on thyroid disruption and developmental neurotoxicity. A systematic literature review has been conducted for these effects (Scopus, Pubmed, Embase), and relevant studies were selected by title/abstract screening (RAYYAN) and full‐text examination. Selected studies were assessed for reliability (SciRAP), and all relevant data were extracted into a database and assessed by Weight of Evidence (WoE) approach. The initial analysis showed potential endocrine adverse effects and endocrine activity, meeting the ED criteria. The use of mechanistic and alternative methods enhanced the outcomes of WoE assessment. Also, the study provides a great hands‐on experience with the most up‐to‐date development in the area of risk assessment and EDs.