Blooms of Ostreopsis spp. have been recently reported along the Mediterranean coasts of Spain, France, Italy, and Greece posing serious risks to human health. Occurrence of Ostreopsis spp. may result in palytoxin contamination of seafood and, in order to prevent sanitary risks, the need exists to develop efficient extraction procedures to be coupled to rapid and sensitive monitoring methods of palytoxin-like compounds in seafood. In the present study, the best conditions for both extraction of palytoxin from seafood and palytoxin quantification by using liquid chromatography tandem mass spectrometry (LC-MS/MS) were investigated. Three seafood matrices (mussels, sea-urchins, and anchovies) were selected and five different extraction systems were tested, namely: the official protocol for extraction of lipophilic toxins and various aqueous methanol or acetonitrile solutions (MeOH/H(2)O 1:1, MeOH/H(2)O 8:2, MeCN/H(2)O 8:2 and MeOH 100%). Extraction with MeOH/H(2)O 8:2 provided the best results in terms of accuracy and matrix interference on LC-MS/MS detection of palytoxin. Accuracy and intra-day reproducibility (n = 3) were evaluated for all the selected matrices but only for mussels at three spiking concentration levels, including the provisional limit proposed by the Community Reference Laboratory for marine biotoxins (250 μg kg(-1)). Limits of quantitation of palytoxin in mussels, sea-urchins and anchovies tissues were calculated using matrix-matched standards; taking into account extraction efficiency of MeOH/H(2)O 8:2, they resulted to be 228, 343, and 500 μg kg(-1), respectively.