The reproducibility of a metabolomics method has been assessed to identify changes in tumour cell metabolites. Tissue culture media extracts were analyzed by reverse phase chromatography on a Waters Acquity T3 column with a 13 min 0.1% formic acid: acetonitrile gradient on Agilent and Waters LC-Q-TOF instruments. Features (m/z, RT) were extracted by MarkerLynx (Waters) and Molecular Feature Extractor (Agilent) in positive and negative ionization modes. The number of features were similar on both instruments and the reproducibility of ten replicates was <35% signal variability for approximately 50% and 40% of all ions detected in positive and negative ionization modes, respectively. External standards spiked to the matrix showed CVs <25% in peak areas within and between days. U87MG glioblastoma cells exposed to the PI 3-Kinase inhibitor LY294002 showed significant alterations of several confirmed features. These included glycerophosphocholine, already shown by NMR to be modulated by LY294002, highlighting the power of this technology for biomarker discovery.