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Microsampling allows the collection of blood samples using a method which is inexpensive, simple and minimally-invasive, without the need for specially-trained medical staff. Analysis of whole blood provides a more holistic understanding of per- and polyfluoroalkyl substances (PFAS) body burden. Capillary action microsamplers (Trajan hemaPEN®) allow the controlled collection of whole blood as dried blood spots (DBS) (four 2.74 µL ± 5 %). The quantification of 75 PFAS from DBS was evaluated by comparing five common extraction techniques. Spiked blood (5 ng/mL PFAS) was extracted by protein precipitation (centrifuged; filtered), acid-base liquid-liquid extraction, trypsin protease digestion, and weak anion exchange (WAX) solid-phase extraction with analysis by high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Filtered protein precipitation was the most effective extraction method, recovering 72 of the 75 PFAS within 70 to 130 % with method reporting limit (MRL) for PFOS of 0.17 ng/L and ranging between 0.05 ng/mL and 0.34 ng/mL for all other PFAS. The optimised method was applied to human blood samples to examine Inter- (n = 7) and intra-day (n = 5) PFAS blood levels in one individual. Sixteen PFAS were detected with an overall Σ 16 PFAS mean = 6.3 (range = 5.7-7.0) ng/mL and perfluorooctane sulfonate (branched and linear isomers, ΣPFOS) = 3.3 (2.8-3.7) ng/mL being the dominant PFAS present. To the authors knowledge, this minimally invasive self-sampling protocol is the most extensive method for PFAS in blood reported and could be a useful tool for large scale human biomonitoring studies., Competing Interests: Declaration of Competing Interest Human blood was voluntarily self-sampled with informed consent by a member of the research cohort, in compliance with The University of Melbourne Research Ethics and Biorisk Management Policy (MPF1341) and the Australian Government National Statement on Ethical Conduct in Human Research (2007). Ethics approval was gained through the University of Melbourne STEMM 1 Human Research Ethics Committee (2023-25725-44910-3). The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)