In this work, the full optimisation and validation procedure to analyse a wide set of emerging organic contaminants in biotissues (mussel and fish muscle, liver, gills and brain) and biofluids (fish plasma and bile) is described. The target families include artificial sweeteners, industrial products, hormones, pharmaceutical and personal care products, pesticides and phytoestrogens. Different clean-up strategies (hydrophilic-lipophilic-balanced (HLB) solid-phase extraction, Florisil solid-phase extraction and liquid-liquid extraction followed by HLB solid-phase extraction and microextraction based on polyethersulfone polymer) were evaluated for the clean-up of focused ultrasonic solid-liquid extraction (FUSLE) extracts before the analysis by liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). The methods afforded satisfactory apparent recovery values (71-126%) using isotopically labelled analytes and matrix-matched calibration approach, regardless of the matrix. Method detection limits in the range of 4-48 ng/g and 0.3-111 ng/L were obtained for biotissues and biofluids, respectively. The developed method was applied to determine the uptake and tissue distribution in juvenile gilt-head bream (Sparus aurata) during 7 days in seawater, and unexpectedly, perfluoro-1-butanesulfonate tended to accumulate in liver and, to a lesser extent, in muscle and gills. Furthermore, real mussel samples collected in the Basque coast were also analysed and the presence of the highly consumed valsartan (7 ng/g) and telmisartan (6.8 ng/g) compounds in bivalves is reported for the first time here. Graphical abstract ᅟ.