AbstractRecent advances in liquid chromatography and mass spectrometry have made it possible to increase the throughput of phytochemical analyses at high sensitivity. These improvements have made it more feasible to monitor metabolic processes at the metabolite level. Glucosinolates, the primary defense compounds in the model plant Arabidopsis thaliana, in particular, have gained increasing attention as model compounds for quantitative genetics and metabolic regulation. Depending on the plant species, tissue, glucosinolate content, complexity of the glucosinolate profile, and, most importantly, the overall purpose of the experiment, different choices need to be made regarding the methods of extraction and analysis. In this chapter, we describe different approaches for the analysis of glucosinolates and highlight advantages, disadvantages, and technical pitfalls. © 2016 by John Wiley & Sons, Inc.