Here we describe a new method of glioma cell visualization in living brain slices that can be used for evaluation of tumor size or visualization of internal tumor structures. Glial cells, as well as glioma cells of glial origin, express high levels of organic cation transporters. We demonstrate that application of a fluorescent substrate for these transporters 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+) to the incubation medium leads to quick accumulation of fluorescence in glioma cells during early developmental stages and in astrocytes, but not in neurons. Stained brain slices can be immediately investigated using confocal or fluorescence microscopy. Glioma and glial cells can be discriminated from each other because of their different morphology. The method described has the advantage of staining living tissue and is simple to perform.