A first cousin of Oliver Cromwell, Hampden was educated at the University of Oxford and the Inner Temple, London, and entered the House of Commons in 1621. There he quickly became a specialist in matters of taxation and a close friend of Sir John Eliot, a leading Puritan critic of the crown. In 1627 Hampden was imprisoned for nearly a year for refusing to contribute a forced loan demanded by the king. When Eliot died in 1632, after three years in prison, Hampden's ill will for Charles was firmly established.

Hampden resisted on principle the payment of ship money, a levy collected by the king for outfitting his navy. Only Parliament was empowered to levy taxes, however, and Hampden reasoned that, as Parliament could meet only when summoned by the king, Charles was, in effect, eliminating the need to call Parliament if he could impose taxes himself. The king contended, however, that ship money was a type of tax that by custom did not need the approval of Parliament. In 1635 Hampden refused to pay 20 shillings in ship money, and the case went before the 12 judges of the Court of the Exchequer. Although seven of the judges upheld Charles and five sided with Hampden (1638), the narrow majority received by the king may have been a factor that encouraged widespread resistance to the tax.

During the Long Parliament, which convened in November 1640, Hampden became the principal lieutenant of Parliamentary leader John Pym in a vigorous attack on royal policies, and he was one of the five members who successfully evaded arrest by the king in January 1642. After the outbreak of the Civil War between the Parliamentarians and the Royalists in August 1642, Hampden served as a colonel in the Battle of Edgehill, Warwickshire (October), but on June 18, 1643, he was mortally wounded in a skirmish with Royalists at Chalgrove Field, near Thame.