Metabolomics aims to systematically gather (quantitative) information on metabolites in the cell and is commonly viewed as the "missing link" between genomics, transcriptomics, and physiology. Typical metabolomics platforms consist of two main steps, the quenching and extraction of metabolites from plant material and the (un) targeted quantification of the extracted metabolites. Brachypodium distachyon, a native grass species of the Mediterranean region, is an attractive model plant to study temperate crops. In recent years, despite an increasing interest in genomic and transcriptomic studies, metabolomics studies for B. distachyon are still in their infancy. Drought, an abiotic stress factor, causes severe loss in plant productivity and it is therefore crucial to understand its effect on plant metabolism, in particular its metabolome. The objective of this study is to set up a quantitative plant metabolomics platform for B. distachyon to evaluate and optimize alternative metabolite extraction protocols (methanol and methanol-chloroform extraction). Focusing on three reporter metabolites (ATP, glucose, and starch) for metabolite quantification and two extraction protocols, we compared the drought stress response of 2 different genotypes from different geographical regions of Turkey. Results revealed that there is no "one-protocol-fits-all", pointing to significant differences in metabolite recoveries upon different extraction protocols, though methanol extraction generally yielded higher recoveries. Drought stress resulted in higher glucose levels while ATP and starch levels did not show statistically significant or consistent changes. In conclusion, the impact of drought stress on B. distachyon metabolism was significant and this study could be the basis for further metabolomics studies on plant stress response.