Given the nature of medieval artifacts and resulting research requirements, a precise temporal classification is essential. It is especially important for the purposes of medieval archaeology in interpreting archaeological finds/finding situations and identifying them with a historical events or figures, for example, to identify skeletal remains of a known historical figure or to establish a chronological sequence of various cultural and architectural changes within an area. Due to the fact that the uncertainties of radiocarbon (14C) analyses have been decreasing in recent years, the applicability of 14C dating for such purposes is now growing. In this work, we aim to demonstrate the current possibilities of the use of AMS 14C analyses on specific cases and confront the results with other available data. 14C data from skeletal remains of members of the oldest Czech ruling dynasty of the Přemyslids (about 880–1306 AD) were obtained in recent years. Archaeological research conducted in the three oldest churches in the Prague Castle discovered skeletal remains of three members of the second, two members of the fourth and two members of the fifth generation. This case study of the application of 14C data has three parts: i) identification of excavated individuals; ii) demonstration of the application using current AMS-based analysis of 14C on medieval osteological material and tests of our preparation method; iii) contributing to discussion and consulting with other problematical 14C age alteration influenced by diet, age of bone collagen or seasonal variation of 14C activity. The obtained results and the issues arising from them clearly highlight the necessity of a multidisciplinary cooperation in this type of study.