He was born in West Pembroke, Maine, USA of Canadian parents.
While a 22-year-old student studying medicine at the University of Toronto, he worked as an assistant to Dr. Frederick Banting and played a vital role in the discovery of the pancreatic hormone insulin, one of the most significant advances in medicine at the time. Dr. Banting would share his 1923 Nobel Prize in Medicine with Charles Best.
During World War II (1939-1945) Dr. Best was influential in the establishing of a Canadian program for securing and usage of dried human blood serum and in his later years he would act as adviser to the medical research committee of the United Nations World Health Organization.
Dr. Best is interred in Mount Pleasant Cemetery, Toronto, Ontario, Canada.