Unlike other handbooks in this emerging field, this guide focuses on the challenges and critical parameters in running a metabolomics study, including such often-neglected issues as sample preparation, choice of separation and detection method, recording and evaluating data. By systematically covering the entire workflow, from sample preparation to data processing, the insight and advice offered here helps to clear the hurdles in setting up and running a successful analysis, resulting in high-quality data from every experiment. Based on more than a decade of practical experience in developing, optimizing and validating metabolomics approaches as a routine technology in the academic and industrial research laboratory, the lessons taught here are highly relevant for all systems-level approaches, whether in systems biology, biotechnology, toxicology or pharmaceutical chemistry.