<p>Per- and polyfluoroalkyl substances (PFAS) have attracted increasing concern due to their environmental persistence and potential adverse health effects. In this study, a high-performance liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of 56 PFASs in human plasma. After the plasma sample was extracted with methanol, 5 mg primary secondary amine (PSA), 20 mg graphitized carbon black (GCB), and 30 mg C18 were added for QuEChERS purification, followed by nitrogen drying and reconstitution. The chromatographic separation was performed on a C18 column with a gradient elution and was quantified on mass spectrometry with electrospray ionization in negative mode. The method was validated and good linearity was obtained in the range of 0.050–50.0 ng/mL. The limits of detection were 0.0024 to 2.05 ng/mL, and except for a few perfluoroalkyl phosphinic acids and fluorinated polymer-based substances with low signal intensities, most PFASs showed satisfactory recoveries of 70–120%, with relative standard deviations below 12%. The validated method was applied to analyze plasma samples from pregnant women and males over 50 years old. The results revealed widespread PFAS contamination in the population, with perfluoroalkyl carboxylic acids as the dominant contributors, followed by perfluoroalkyl sulfonic acids and accompanied by a limited number of emerging PFAS alternatives. There are population-specific differences in PFAS exposure, with median concentrations typically higher in males aged over 50 years than in pregnant women, but 6:2 FTS is much higher in pregnant women. The established method is sensitive and efficient, and the results highlight the importance of population-stratified biomonitoring of PFASs.</p> Graphical abstract <p></p>