Beginning in the 16th century, the Isthmus of Panama was identified as a region of strategic importance. Although mountainous and prone to adverse weather, it provided the most direct route between the Atlantic and Pacific oceans. During this period the Isthmus served as the primary route for the shipments of silver and gold extracted from the mines of Peru. The bullion was transported via coastal armadas from Peru to Panamá la Vieja, where it was loaded onto pack mules and hauled across the Isthmus to Nombre de Dios until 1597, and after that to Portobelo. Once the bullion arrived at the Caribbean port cities it was transferred to the ships of the Armada de la Guardia de la Carrera de Indias and shipped across the Atlantic to the royal coffers of Spain. Because of the Isthmus’s role in the transportation of valuable commodities, it quickly became a region prone to attacks by pirates and privateers looking to profit from the plunder of Spanish assets. Thus the Spanish crown began a campaign to defend the Isthmus early on and repeatedly adapted its defensive strategy in order to meet the ever-changing tactics of the pirates and privateers. This thesis investigates the history of the Isthmus of Panama and the ways in which Spain defended this strategically significant locale during the 16th century. It incorporates an historical analysis of the tactics planned, ordered, and executed by the crown; an overview of the most relevant structural remains of the fortifications built during this period; and a synopsis of previous archaeological investigations, as well as the prospects of future archaeological research. The thesis begins with a brief history of the three main cities located on the Isthmus as well as a general description of the geography and climate in order to better explain the challenges faced by the inhabitants, soldiers, and attackers in this region. It then discusses the strategic importance of the Isthmus as it was perceived in the 16th century. Since defense would not have been necessary had it not been for the presence of pirates and privateers, the history of attacks on the Isthmus is discussed, and a general overview of piracy in the Spanish Main during the period under analysis is presented. Lastly, the archaeological work previously undertaken in the region is examined and summarized, and recommendations for further research are provided in an effort to provide a basis for future study of the ships and infrastructure used during this era for the defense of the Isthmus of Panama.