The Joint Research Centre, in cooperation with the Commissariat à l’Energie Atomique et aux Energies Alternatives, produced a novel 243Am spike reference material for mass spectrometry. Americium solution with an isotopic composition of 88% 243Am and 12% 241Am was used as the source for the preparation of the spike material. The certified value of 5.696 (11) nmol g−1 for the amount content of 243Am and 0.136138 (54) for the n(241Am)/n(243Am) amount ratio were assigned. The assigned values from mass spectrometry were confirmed by alpha-particle spectrometry, alpha-particle counting at a defined solid angle, and high-resolution gamma-ray spectrometry. Furthermore, an external validation of the certified values was obtained from the results of an interlaboratory comparison exercise, using this americium reference solution as the test sample.