He is known for introducing starch as a medium for gel electrophoresis in 1955,[3] and for the discovery, simultaneously with Mario Capecchi and Martin Evans, of the technique of homologous recombination of transgenic DNA with genomic DNA, a much more reliable method of altering animal genomes than previously used, and the technique behind gene targeting and knockout mice.

[9] In 1951, he received a Master of Arts degree and a Doctor of Philosophy in biochemistry under Ogston's supervision; his thesis was entitled "Physico-chemical properties of solutions of proteins".

[12][1] Smithies was awarded a Commonwealth Fund fellowship to take up a post-doctoral position in the United States, in the laboratory of J. W. Williams at the University of Wisconsin–Madison's Department of Chemistry.

[16] Smithies developed the technique of gel electrophoresis using a starch matrix, as a sideline of (unproductive) research into an insulin precursor molecule, at the University of Toronto.

He used starch electrophoresis to reveal differences between normal human plasma proteins, and in collaboration with Norma Ford Walker, showed that the variation was inherited, which stimulated his interest in genetics.

[14][17] This research is the basis of methods used worldwide to investigate the role of particular genes in a wide range of human diseases including cancer, cystic fibrosis and diabetes.

[18] In 2002, Smithies worked with his wife, Nobuyo Maeda, studying high blood pressure using genetically altered mice.

[20] He won the 2007 Nobel Prize in Physiology or Medicine, jointly with Capecchi and Evans, "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells.