This paper aims to cover the main strategies based on ion mobility spectrometry (IMS) for the analysis of biological samples. The determination of endogenous and exogenous compounds in such samples is important for the understanding of the health status of individuals. For this reason, the development of new approaches that can be complementary to the ones already established (mainly based on liquid chromatography coupled to mass spectrometry) is welcomed. In this regard, ion mobility spectrometry has appeared in the analytical scenario as a powerful technique for the separation and characterization of compounds based on their mobility. IMS has been used in several areas taking advantage of its orthogonality with other analytical separation techniques, such as liquid chromatography, gas chromatography, capillary electrophoresis, or supercritical fluid chromatography. Bioanalysis is not one of the areas where IMS has been more extensively applied. However, over the last years, the interest in using this approach for the analysis of biological samples has clearly increased. This paper introduces the reader to the principles controlling the separation in IMS and reviews recent applications using this technique in the field of bioanalysis.