Introduction: Polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) belong to a well-known group of pollutants. Present in feedstuffs, they bioaccumulate in tissues of food-producing animals. Food is the source of over 90% of human PCDD/Fs and DL-PCBs intake. Thus, feed control is one of the pillars of the EU strategy and a mean of reducing human exposure. The article presents AhR based reporter gene bioassay method for PCDD/Fs and DL-PCBs analysis in feed and its validation results. Material and Methods: Analytes were extracted from samples with fat. Subsequently, fat and other interferences were removed from extract using sulphuric acid modified silica. Extract was further cleaned and PCDD/Fs separated from DL-PCBs using carbon column. Contaminants detection was performed using H1L6.1c3 cell line, which produces luciferase in response to AhR ligands present in extract. Results: Performance characteristics (repeatability, reproducibility, and apparent recovery) fulfil the requirements of Regulation 2017/771/EU. The positive correlation between bioassay and reference HRGC-HRMS method was confirmed. Moreover, the role of screening method used in connection with the confirmatory HRGC-HRMS method in providing feed and food safety has been discussed. Conclusion: Bioassay is a useful method for dioxin and DL-PCBs analysis, allowing cost reduction of monitoring programmes with minimal risk of false negative results.