Best, who was born in West Pembroke, Maine, graduated in physiology and biochemistry from the University of Toronto in 1921. In the summer of that year he gave up a lucrative holiday playing professional football and baseball to begin work with Frederick Banting. Together they isolated the hormone insulin, and showed its use in the treatment of diabetes mellitus. Banting was furious when Best was not awarded a share in the 1923 Nobel Prize for physiology or medicine for the discovery of insulin.

Best remained at the University of Toronto and gained his MB in 1925. He was made head of the physiology department in 1929 and became director of the Banting and Best Department of Medical Research when Banting was killed in 1941. He continued the work on insulin throughout these years and in an important paper published in 1936 he suggested the administration of zinc along with insulin to reduce its rate of absorption and make it more effective over a longer time. He also studied cardiovascular disease and established the clinical use of heparin as an anticoagulant for blood in the treatment of thrombosis. He discovered the vitamin choline, which prevents liver damage, and the important enzyme histaminase, which takes part in local inflammation reactions, breaking down histamine.