An important procedure in biomedical research is the detection of genes that are differentially expressed under pathologic conditions. These genes, or at least a subset of them, are key biomarkers and are thought to be important to describe and understand the analyzed biological system (the pathology) at a molecular level. To obtain this understanding, it is indispensable to link those genes to biological knowledge stored in databases. Ontological analysis is nowadays a standard procedure to analyze large gene lists. By detecting enriched and depleted gene properties and functions, important insights on the biological system can be obtained. In this chapter, we will give a brief survey of the general layout of the methods used in an ontological analysis and of the most important tools that have been developed.