We demonstrate for the first time the application of an endoscopic fluorescence lifetime imaging microscopy (FLIM) system to the intraoperative diagnosis of glioblastoma multiforme (GBM). The clinically compatible FLIM prototype integrates a gated (down to ) intensifier imaging system with a fiber-bundle (fiber image guide of diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, and field of view) to provide intraoperative access to the surgical field. Experiments conducted in three patients undergoing craniotomy for tumor resection demonstrate that FLIM-derived parameters allow for delineation of tumor from normal cortex. For example, at wavelength band emission corresponding to NADH/NADPH fluorescence, GBM exhibited a weaker florescence intensity (35% less, -value ) and a longer lifetime than normal cortex (-value ). Current results demonstrate the potential use of FLIM as a tool for image-guided surgery of brain tumors.