The gap of the post-genomic era is increasingly being filled by the metabolomics approach, comprising a technology for analyzing small molecule endogenous metabolites (≤1500 Dalton) in complex biological samples. This new analytical science has progressed within the last years particularly with regard to improvements in mass spectrometry based detection, now allowing highly robust, reproducible, selective and sensitive qualitative or quantitative analysis of endogenous metabolites. The precise and accurate quantitation of these metabolites via targeted metabolomics, now critically contributes to the quantitative analysis of endogenous compounds in biomarker discovery and validation thus to future personalized therapy. The analytical methods of choice in (MS-based) targeted metabolomics primarily are HPLC-API-MS/MS, FIA-APIMS/ MS and GC-MS. In the parent paper, we provide an introduction and brief survey on the technological basis of targeted metabolomics in biomarker research, discuss various relevant analytical aspects in mass spectrometry including comparison to non-targeted approaches, effects of sample preparation, impact of sample stability, carryover- and matrix effects, need for standardization and for proficiency tests, standardization of analytical methods as well as the requirement for method validation.