High-throughput screening of large collections of plants, whether in the context of gene function analysis, quality trait selection, or metabolic engineering requires robust and rapid methodologies that provide maximum information with minimum sample pre-fractionation. Here, we present a protocol for high-throughput plant metabolomic analysis developed for Arabidopsis and generally applicable to plant green tissue, including other Brassicaceae. The methodology uses combined, flow injection electrospray mass spectrometry (FI-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy analysis. The protocol covers all steps of the process including sample extraction, data acquisition, data processing, and multivariate statistical analysis.