In conclusion, our results demonstrate that Probe 10 is the first ALDH1A3 selective fluorescent tool that is able to preferentially bind to the target enzyme, without inducing significant cytotoxic effects, both in vitro, in human Glioblastoma cells U87MG, 3054, 3060 and in vitro and in vivo murine GL261 cells outlining the tumor growing in the left striatum and invading the adjacent areas of the brain. Here, ALDH1A3 is linked to glioblastoma.