Orthotopic xenograft studies strongly promote the development of personalized and targeted cancer therapies to increase the still very poor life expectancy of glioblastoma (GBM) patients. As the tumor microenvironment (TME) plays an active role in cancer progression, the TME has increasingly become subject of investigations recently.We aimed to assess the metabolome from interstitial fluid (ISF) collected with Open Flow Microperfusion (cOFM) from the TME and compare it to the metabolome from cerebrospinal fluid (CSF). This was done via atraumatic access that has been generated by inoculating mice with human GBM xenograft cells with a previously implanted cOFM probe, and by allowing a xenograft GBM to develop at the interface betwwen the cOFM probe and the surrounding brain tissue.Tumor growth was monitored with magnetic-resonance-imaging. On day 18 post-inoculation, ISF from TME was collected with cOFM and blood, CSF and brain tissue were collected from the same animals at terminal time point. The collected samples were analyzed using a UHPLC–HRMS/MS metabolomics platform. Metabolomics data from GBM animals were analyzed and compared with that from non-GBM animals (controls).We found 1618 metabolic features in the ISF of TME. TME metabolome showed distinct metabolic patterns when compared to normal brain tissue. GBM relevant metabolites such as Kynurenine, Carnosine or Guanidinoacetate exhibited different levels in GBM versus non-GBM tissue in ISF but not in CSF samples. This suggests that metabolomics data obtained from ISF are better suited to provide a picture of the condition within the TME than data from CSF.