Isabel Hampton was born in Welland, Ontario, on August 26, 1859. At 17, she became a public school teacher in Merritton, Ontario. She attended a collegiate institution after high school, but a significant part of her early education was attained through independent study. Hampton enrolled in the Bellevue Hospital Training School for Nurses in 1881 and graduated in 1883. After graduation, she briefly worked as a nurse in New York and later went to work in Rome at St. Paul's House. Here, she worked for a hospital that served American and European travelers.