Liquid chromatography coupled to tandem mass spectrometry (LC-MS) has been used in the last two decades, in drug discovery or drug development as well as in therapeutic drug monitoring (TDM). Due to its high sensitivity and selectivity it is the method of choice for the quantification of drugs and metabolites in biological fluids. More and more the technique is also applied for multi-analytes quantification to support metabolomics studies. To improve sample throughput in TDM a dual Ultra High Performance Liquid Chromatography – Mass Spectrometry assay was developed and validated for the quantification of eight protease inhibitors in plasma and blood. Based on the same platform with two different chromatographic systems (pH 2.7 and pH 6.6) and the addition an automated on-line sample preparation step an assay method could be developed for the analysis of eighty-seven metabolites in plasma and blood. Finally the downsizing of the LC was investigated with the emphasis to improve the sensitivity of LC-MS assay when microsamples are available.