The LSMS group research covers the investigation of all analytical processes from sample collection and preparation to quantification or characterization of compounds of biological interest (metabolites, peptides, proteins, pharmaceuticals). Separation methods hyphenated to Mass Spectrometry are central and support all these method developments. In a context of large cohorts of samples analysis and disease biomarker discovery, multiplexed and fast analyses are a major research focus. To address these needs, the potential of column switching and fundamentals in ion mobility spectrometry are thoroughly explored. This thesis work is dedicated to the development of innovative analytical platforms for low and high molecular weight compounds of interest in clinical or pharmaceutical research. Differential ion mobility spectrometry takes a central part in this work, supporting different types of analytical methods, for the analysis of endogenous and exogenous analytes in various biological matrices (blood, plasma, urine, tissues).