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Phomopsin A (PHO-A), a potent mycotoxin produced by the pathogenic fungus Diaporthe toxica, is a growing international concern due to the high toxicity and prevalence in lupin plants, a vital source of food and feed. This hexapeptide mycotoxin has raised health concerns for both animals and humans, prompting regulatory bodies to set maximum allowable levels in lupin products.Different analytical methods have been developed for PHO-A detection, including Enzyme-Linked Immuno-Sorbent Assay (ELISA) and high-performance liquid chromatography (HPLC) coupled with different detectors; they often lack sensitivity and selectivity required to meet maximum regulatory limits (MRL). In this work, we present a robust and sensitive method for PHO-A quantification and determination, developed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).This method incorporates a selective clean-up step using Micro Solid Phase Extraction (µ-SPE) to efficiently purify samples, ensuring a rapid, efficient, and selective recovery of PHO-A from the complex lupin matrix. The clean-up step not only helps in achieving lower detection limits but also minimizes matrix effects and contamination. The proposed method was validated following international guidelines, demonstrating reliable recovery, matrix effects, linearity, accuracy, and precision.Additionally, this method was successfully applied to artificially contaminated Lupinus albus L. samples, confirming the suitability for assessing PHO-A contamination. This analytical approach fills a critical gap in the literature by providing a specific, sensitive, and selective method for PHO-A analysis, contributing to the safety of lupin products and field monitoring.