As a 22-year-old medical student at the University of Toronto he worked as an assistant to Dr. Frederick Banting and played a major role in the discovery of the pancreatic hormone insulin—one of the most significant advances in medicine, enabling an effective treatment for diabetes.
In 1923, the Nobel Prize committee honoured Banting and J.J.R. Macleod with the Nobel Prize in Medicine for the discovery of insulin, ignoring Best. This incensed Banting, who voluntarily shared half of his award money with Best.
Best succeeded Macleod as professor of physiology at University of Toronto in 1929. During World War II he was influential in establishing a Canadian programme for securing and using dried human blood serum. In his later years, Best was an adviser to the medical research committee of the United Nations World Health Organization.
In 1967 he was made a Companion of the Order of Canada. In 1994 he was inducted into the Canadian Medical Hall of Fame. In 2004, he was inducted into the National Inventors Hall of Fame. Dr. Charles Best Secondary School in Coquitlam, British Columbia, C.H. Best West Elementary School in Burlington, Ontario, and C.H. Best East Middle School in Toronto, Ontario, are named in his honour.
Best married Margaret Hooper Mahon in Toronto in 1924. They had two sons.
Charles Best is interred in Mount Pleasant Cemetery, Toronto.