A methodology combining finger-pricked blood sampling, microwave accelerated fatty acid assay, fast gas chromatography data acquisition, and automated data processing was developed, evaluated and applied to a population study. Finger-pricked blood was collected on filter paper previously impregnated with 0.05 mg of the antioxidant butylated hydroxytoluene and air-dried at room temperature. Transmethylation was accelerated by microwave irradiation in an explosion-proof multimode microwave reaction system. The chemical procedure was based on a one-step direct transmethylation procedure catalyzed by acetyl chloride. The short-term stability of PUFA in blood dried on filter paper and stored overnight at room temperature was examined using venous blood. The recoveries ranged from 97–101 % for the categorized fatty acids as well as the ratios of n-6 to n-3 PUFA and the n-3% highly unsaturated fatty acid. Specifically, recoveries were 99, 98, 97, and 97 % for linoleic acid (18:2n-6), arachidonic acid (ARA), α-linolenic acid (ALA), and docosahexaenoic acid (DHA), respectively. The mol% (mean ± SD, 95% confidence interval) of fatty acid composition in subjects from the population study was determined as 36.2±3.8 (35.8, 36.7), 23.2±3.0 (22.8, 23.5), 36.8±3.5 (36.4, 37.2) and 3.79±1.0 (3.68, 3.91) for the saturated, monounsaturated, n-6 and n-3 PUFA, respectively. Individually, the mean mol% (95% CI) was 22.6 (22.3, 22.9) for 18:2n-6, 9.5 (9.3, 9.7) for ARA, 0.51 (0.49, 0.53) for ALA, 0.42 (0.38, 0.47) for eicosapentaenoic acid (EPA), and 1.67 (1.61, 1.73) for DHA. This methodology provides an accelerated yet high-efficiency, chemically safe, and temperature-controlled transmethylation, with diverse laboratory applications including population studies.