Diarrhetic shellfish poisoning (DSP) is a severe gastrointestinal illness caused by consumption of shellfish contaminated with toxigenic dinoflagellates. The main toxins responsible for DSP are okadaic acid (OA), DTX-1, DTX-2, and DTX-3, the latter being a complex mixture of 7-O-acyl derivatives of the first 3. In this study, existing methods based on liquid chromatography (LC) combined with mass spectrometry (LC-MS) and LC with fluorometric detection (LC-FLD) of anthryldiazomethane (ADAM) derivatives were improved upon to achieve a high degree of accuracy and precision for the determination of DSP toxins in a new mussel tissue reference material (MUS-2). All experimental parameters were examined comprehensively, and a new internal standard and a new solid-phase extraction cleanup method were introduced. Quantitative extraction of DSP toxins from shellfish tissue was achieved by exhaustive extraction with aqueous 80% methanol. Cleanup was accomplished by partitioning the crude aqueous methanol extract with hexane to remove lipids and then with chloroform to isolate the toxins. A further cleanup based on an aminopropylsilica column was useful for LC-MS and looks promising for the ADAM/LC-FLD method. The internal standard, 7-O-acetylokadaic acid, synthesized by partial acetylation of OA, improved accuracy and precision by correcting for incomplete recoveries in extraction, cleanup, and derivatization steps and for volumetric errors and instrumental drift. An improved silica cleanup after ADAM derivatization also was developed by controlling the activities of both sorbent and solvents. The methods were tested with various mussel tissue samples. The resulting improved methods will be useful to analysts involved in routine monitoring of DSP toxins.