The development of a reliable analytical method for determining biotoxins levels in bivalves is important for the risk assessment of sea food. However, analysis of these toxins in high-lipid bivalves has several limitations due to the coexistence of lipid matrices, low biotoxin concentrations, and the different physiochemical properties of the various biotoxins. In this study, UHPLC-MS/MS method combined with an effective cleanup process (freezing-lipid filtration and solid-phase extraction) was developed for reliable quantification of six biotoxins in high-lipid bivalves. To eliminate lipid components after sample extraction, freezing-lipid filtration was applied to achieve lipid removal efficiency of approximately 91%. For further cleanup, a Strata-X solid-phase extraction cartridge was applied to separate the biotoxins from any remaining interfering compounds. Subsequently, trace biotoxins with acidic and neutral properties were determined simultaneously and sensitively by UHPLC-MS/MS using time segment polarity switching. Validation tests using a certified reference material and spiking experiments showed this method to be acceptable, with a relative standard deviation of < 14.44% and limits of quantitation of 0.63-3.08 ng/mL. The developed method was successfully applied to quantify biotoxins in 40 oyster and mussel samples collected from Korean fishery markets, ensuring sea food safety.