Abstract We have developed a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for thyroxine (T 4) in dried blood samples spotted on filter paper. The assay is carried out on microtiter plates without extraction or centrifugation steps. The detection limit of the assay is 5 pg/disc/well, equivalent to 1.25 μg/1 of whole blood or 2.5 μg/1 of serum. Intra- and inter-assay coefficients of variation for various T 4 concentrations are 2.4–9.0% and 5.9–17.5% respectively. Correlation between the proposed ELISA method and the RIA is good ( r = 0.900, n = 62, y(RIA) = 0.99 x(ELISA)+9.90). The ELISA method is useful for mass-screening of neonatal congenital hypothyroidism using dried blood samples on filter paper, is very simple and one person can assay more than 300 samples per day.