This thesis describes novel strategies for sample preparation and for ultra-fast, mass spectrometry-based quantitation of drugs and metabolites in biological fluids. A first chapter provides an overview of quantitative bioanalysis, reviewing sample preparation, liquid chromatography (LC) and mass spectrometry (MS) aspects. A second chapter reports a LC-MS method for the sensitive quantitation of oxycodone and three of its metabolites in human plasma, based on column switching for automated sample preparation. In a third chapter, a non-chromatographic assay using matrix-assisted laser desorption/ionization (MALDI) MS is presented as an alternative to the well-established LC-MS approach, and allows to reduce analysis time from several minutes to a few seconds. Whereas plasma is the most used matrix for bioanalysis, chapter four discusses the use of dried blood spots. Chapter five presents a novel tube-based format for DBS collection and analysis, and a new sample preparation strategy associated to it, namely micro cellulose-supported liquid-liquid extraction.