To explore the chemical composition of root exudates of the model plant Arabidopsis thaliana a workflow for nontargeted metabolite profiling of the semipolar fraction of root exudates was developed. It comprises hydroponic plant cultivation and sampling of root exudates under sterile conditions, sample preparation by solid-phase extraction and analysis by reversed-phase UPLC/ESI-QTOFMS. Following the established workflow, root exudates of six-week-old plants were profiled and a set of reproducibly occurring molecular features was compiled. To structurally elucidate the corresponding metabolites, accurate mass tandem mass spectrometry and on-line hydrogen/deuterium exchange were applied. Currently, a total of 103 compounds were detected and annotated by elemental composition of which more than 90 were structurally characterized or classified. Among them, 42 compounds were rigorously identified using an authenticated standard. The compounds identified so far include nucleosides, deoxynucleosides, aromatic amino acids, anabolites and catabolites of glucosinolates, dipeptides, indolics, salicylic and jasmonic acid catabolites, coumarins, mono-, di- and trilignols, hydroxycinnamic acid derivatives and oxylipins and exemplify the high chemical diversity of plant root exudates. CI - Copyright (c) 2014 Elsevier Ltd. All rights reserved.