In 1218, King Alfonso IX of Leon founded the University of Salamanca, which is considered the oldest of the existing Spanish universities. Outstanding among the legislation founding the University of Salamanca is the Charter granted by King Alfonso X, dated 8 May, 1254, which established the rules for organization and financial endowment. Also important are the Papal Bulls of Alexander IV, issued in 1255, which confirmed the founding of the University, recognised the universal validity of the degrees awarded by it and granted it the privilege of having its own seal.

The regulation of studies and academic life fell to the Papacy during the Middle Ages (the constitution of 1411, of Benedict XIII, and that of 1422, of Martin V), and to the Monarch and his Council beginning in the 16th century: the statutes of 1538, 1551, 1561, 1594, 1604 and 1618. These university regulations established certain books, authors and teaching matter and were to be in force until the reforms of the Enlightenment: Roman or Justinian Law in Law, Papal decrees in Canon Law, scholastic metaphysical theology in Theology, Galen and Hippocrates in Medicine, Aristotelian philosophy in Arts-Philosophy, Euclid and Ptolomy in Astrology/Mathematics and the Latin and Greek classics.

Throughout the 15th and 16th centuries, the University of Salamanca took part in the new humanist trends, Nebrija's teaching being a good example. Furthermore, there were a great amount of scientific manuscripts in some of the Colleges, such as the Major College of St. Bartholomew, linked to the Chairs of Natural Philosophy, Mathematics and Astronomy. In the middle years of the 16th century, the coming together of Law, Thomist Theology, the new logics and the classical languages crystallized in the so-called School of Salamanca, represented in the figure of Francisco de Vitoria. Some of his most important contributions were the practical reflection on certain problems derived from European expansion and colonization and American trans-culturation: the nature of power and justice, the rights of the person and the State, international community and the law of nations or peoples, international conflicts and just wars. Together with the predominant official channels of transmission of knowledge, in the different subject matter, other interpretations of knowledge and teaching were added, such as the theological and philological thought of Fray Luis de Leon, which brought complexity and cultural richness to 16th century Salamanca.

The Caroline study plan of 1771 contained stipulations that contributed to the introduction of new approaches and subject matter (property or patrial law, conciliary matters, positive theology, arithmetic, geometry, algebra and experimental physics), although their success was limited. The culmination of the interventionist policy of the monarchy in the University was the study plan approved by Salamanca in 1807, which spread to all the universities under the monarchy, although its application was interrupted by the events of the War of Independence. Moreover, the organizational model of Salamanca spread to the Latin American universities, where it was adapted to the specific circumstances of each institution.

The highest numbers of students were recorded in the 16th century: at the end of the 14th century the institution had 500–600 students, whereas at the beginning of the 16th century there were 2,500 students registered each year, to grow to be 6,500 in the 1580's. The prestige of Salamanca drew to it students from all over the Peninsula, and even Europeans and Spanish Americans in greater numbers than any other Hispanic university of the age. From the 17th century on, university registration began to decline, and in the middle of the 18th century there were only 2,000 students registered.