The presence and accumulation of marine biotoxins, including paralytic shellfish toxins (PSTs) and tetrodotoxin (TTX) analogues, in marine organisms poses a significant food safety concern. This study describes the validation of a quantitative confirmatory HILIC–LC–MS/MS method for the simultaneous determination of a broad panel of marine toxins in four matrices: mussels, clams, sea snails, and sea urchins. The method covered 17 target compounds, including TTX analogues (TTX, 4-epiTTX, and 4.9-anhTTX), saxitoxin and analogues (STX, dcSTX, NEO, and dcNEO), gonyautoxins (GTX 1–4, GTX 2–3, GTX 5, GTX 6, and dcGTX 2–3), and sulfocarbamoyl toxins C1–C2. Particular attention was devoted to the evaluation of matrix effects (ME), a critical factor affecting LC–MS/MS quantification. The method was validated according to SANTE/11312/2021 guidelines, showing satisfactory selectivity, precision (RSD ≤ 20%), and accuracy (recoveries within 70–120%). Significant matrix-dependent signal suppression and enhancement were observed among the investigated species. Matrix-matched calibration enabled reliable quantification in mussels, clams, and sea snails, whereas sea urchin samples showed greater analytical complexity. The validated method provides a robust approach for the determination of PSTs and TTX analogues in different marine organisms and supports its application in official monitoring programs.