a b s t r a c tA rapid and fully automatic method for determining 21 per-and polyfluoroalkyl substances (with carbon chains ranging from C4 to C18, including 13 PFCAs, 5 PFSAs, 2 Cl-PFESAs, and PFOSA) in human serum samples was developed. The HPLC parameters, Turboflow column, mobile phase, sample injection volume, loading flow rate, and sample cleanup and elution time were optimized. 25 L serum sample was directly injected into the developed on-line Turboflow SPE HPLC-MS/MS system for analysis after dilution. Matrix effects were corrected due to the matrix removal efficiency of the Turboflow column and sufficient types of internal isotope standards that were used. The established method showed a good linearity (r 2 > 0.99), rapid processing time (20 min per sample), satisfactory recoveries (matrix spiked recoveries range from 84.6% to 114%) and precision (intra-day and inter-day RSDs ranged from 1.5% to 9.2% and from 1.1% to 7.0%, respectively). The limits of detection (LODs) of the 21 analyzed PFASs were between 0.008 and 0.19 ng mL −1 . The LODs of short-and long-chain PFASs, such as PFBA, PFPeA, PFHxDA, and PFODA, were 0.008, 0.022, 0.15 and 0.19 ng mL −1 , respectively; the spiked recoveries of these PFASs were 101, 105, 87.1, and 85.8%, respectively. Both the LODs and recoveries were better than previous studies. Further, serum PFASs concentrations detected by the presented on-line SPE method were consistent with the traditional off-line SPE method (r: 0.98-0.99), which verified the accuracy and applicability of the present method. The method shows good practical prospects in the analysis of trace per-and polyfluoroalkyl substances in human serum.