The CALUX bioassay is at present the best screening method for dioxins and dioxin-like (dl) polychlorinated biphenyls (PCBs) in food and feed, and the only assay used in routine monitoring and during larger incidents. Furthermore, the use of bioassays in addition to chemical reference methods allows the discovery of novel contaminants with potentially adverse effects on human health. The CALUX bioassay shows a clear dose-related response both with dioxin standards and contaminated samples, but requires a clear strategy with respect to decision limits, necessary to deal with the many different action and tolerance limits in European Union (EU) legislation. In future, the CALUX bioassay will profit from further optimization, especially with respect to the clean-up procedure. As demonstrated in the limited number of interlaboratory studies performed so far, this should lead to an even more robust assay that can be easily introduced into less experienced laboratories. The present article discusses the different issues, based on some practical examples from the EU-DIFFERENCE project and gives recommendations for future studies.