Yessotoxins (YTXs) are polycyclic ether compounds produced by phytoplanktonic dinoflagellates and accumulated in filter-feeding shellfish. Mouse bioassay is still the official method to detect these toxins, even if it is lacking of specificity and sensitivity. Moreover, there is growing resistance against the use of animal experiments. Many efforts have been made to determine YTXs with other methods. The detection of YTX using a functional assay allows its quantification with an automated and repetitive technique at concentrations in the range of the 1 mg of YTX equivalent/kg European regulatory limit. In this study, an in vitro functional assay based on YTX treatment of MCF-7 cells and resulting in the accumulation of a 100-kDa fragment of E-cadherin was developed on samples of Mytilus galloprovincialis collected from the Adriatic Sea, Italy, along the coasts of Abruzzo, Molise, and Emilia Romagna regions. The YTX concentrations ranged from 0.2 to 1.8 mg of YTX equivalent/kg. The occurrence of levels exceeding the above mentioned limit was observed only in samples of Emilia Romagna region. This last result could represent a risk for human health, but these shellfish were not intended to consumers, because they belonged to a preventive monitoring program.