Decision support systems (DSSs), as means of diffusing clinical guidelines, are powerful software system that will result in an improvement of medical practices. However, they are not invariably efficient and may suffer from limitations among which are lack of flexibility and weaknesses in the integration of many clinical guidelines for the management of patient's details. Recent research efforts resulted in a vital range of semantic reference systems enriched with vocabularies, thesauri, terminologies, and ontologies. The intensive use of ontologies is included in a new approach to create modern intelligent systems, reusing and sharing pieces of declarative information that plays a significant role in a DSS. A lot of effort has been made to produce standard ontologies for medical knowledge representation. This chapter brings an overview of semantic knowledge representation frameworks such as RDF and OWL for developing ontology and presents a DSS that is enabled by ontology for healthcare domain. A clinical use case is illustrated highlighting the role of ontology in medical DSS. © Springer Nature Singapore Pte Ltd. 2018. All rights reserved.