Combining mass spectrometric tools, a total of 47 in vitro metabolites of okadaic acid (OA), dinophysistoxins 1 and 2 (DTX1 and DTX2), yessotoxin (YTX), azaspiracid1 (AZA1), and pectenotoxin 2 (PTX2) could be detected and confirmed after an incubation with rat liver S9-mix. In a first step, liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS) was used as a screening tool for the identification of in vitro metabolites of lipophilic marine biotoxins. Metabolic phase I and phase II reactions were screened for metabolites by calculating and subsequently monitoring theoretical MS transitions. In a second step, metabolites were confirmed by determination of accurate masses using high resolution MS provided by Orbitrap technology. Subsequently, product ion spectra, precursor ion spectra, and MS3 spectra were recorded for structure elucidation of metabolites. While all investigated toxins were found to form various oxygenated metabolites during the oxidative phase I metabolism, those metabolites varied in the number of added oxygen atoms and in the number of individual isomers. No hints were obtained concerning the formation of glutathione adducts, and a conjugation with glucuronic acid was detected for AZA1 only.