Robert Alexander Stewart Macalister was born in Dublin, Ireland and studied at Cambridge University. Along with Frederick J. Bliss, he excavated several towns in the Shephelah region of Ottoman Palestine from 1898 to 1900. Using advances in stratigraphy building on the work of Flinders Petrie, they developed a chronology for the region using ceramic typology. Upon Bliss' retirement, Macalister became director of excavations for the Palestine Exploration Fund (PEF) in 1901.
From 1902 to 1909 he was responsible for the excavations at Gezer. This was one of the earliest large-scale scientific archaeological excavations in the region. The Gezer calendar found there is a very early paleo-Hebrew calendrical inscription.
Macalister left the field of Biblical archaeology in 1909 to accept a position as professor of Celtic archaeology at University College Dublin, where he taught until his retirement in 1943. During this period, he worked at the ancient Irish royal site at the Hill of Tara and was responsible for editing the catalogue of all known ogham inscriptions from Great Britain and Ireland.
