Anne Hathaway's documented adult life centered on marriage, children and the Shakespeare household. She married William Shakespeare in 1582, and their daughter Susanna was baptized in May 1583. Twins Hamnet and Judith followed in February 1585. Hamnet died in 1596, aged eleven. Parish and property records establish these family events more firmly than later stories about the couple's feelings or the quality of their marriage. No separate paid profession is securely documented for Anne.

The family acquired New Place in Stratford-upon-Avon in 1597, making Anne mistress of one of the town's largest houses. A record places her there in 1601. Her husband's theatrical work took him to London, while the family's Stratford home provided continuity for their children and kin. Household management probably involved servants, provisions and domestic production, but the surviving evidence does not establish Anne's exact daily duties or justify presenting her as a documented brewer, textile worker or business partner.

After Shakespeare died in 1616, Anne survived him for seven years. His will specifically left her the second-best bed and its furnishings; that bequest alone cannot establish estrangement. New Place passed to Susanna and John Hall, and Anne is generally understood to have remained with the family there, although her own will and inventory do not survive. She died in August 1623 and was buried beside her husband in Holy Trinity Church, Stratford.