In 1601, Timberlake sailed into the Mediterranean onboard his merchantmen Trojan (also recorded as Troyan), picking up Muslim travelers in Algiers who were bound for Mecca and Tunis.
Finding no luck in his attempts to sell his goods in the city, Timberlake arranged travel for a pilgrimage to Jerusalem accompanied by another Englishman, John Burrell of Middlesbrough.
A Moroccan who had embarked upon Trojan as a passenger in Algiers encountered Timberlake again at Mamre, near Hebron, as part of a large Syrian cavaran.
When the caravan reached Jerusalem, Timberlake identified himself at the Jaffa Gate as a Protestant and an Englishman; he was arrested and accused by the Ottoman authorities of being a spy, who had never heard of England or Queen Elizabeth.
After his return, Timberlake wrote a lengthy letter about his adventures to friends in London; it was published in 1603 as A True and Strange Discourse on the Travailes of Two English Pilgrims.
[citation needed] Timberlake's account is considered to be a vivid glimpse into the history of Palestine and the situation of Christians there during the period of Ottoman rule.
Timberlake's positive depictions of Catholics and Muslims were viewed with suspicion by some in England, and an unflattering caricature of him was printed in an unauthorised version of his account.
Timberlake was buried in the chancel of the St. Peter's Church in Titchfield, as were Southampton and his eldest son, who had both died of disease in the Low Countries in 1624.
[citation needed] One of Timberlake's sons, Francis, emigrated to Virginia where he settled (or, he may have traveled there earlier with his father and stayed to administer his properties).