To investigate the in-situ response to a stress, grapevine leaves have been subjected to mass spectrometry imaging (MSI) experiments. The Matrix Assisted Laser Desorption/Ionisation (MALDI) approach using different matrices has been evaluated. Among all the tested matrices, the 2,5-dihydroxybenzoic acid (DHB) was found to be the most efficient matrix allowing a broader range of detected stilbene phytoalexins. Resveratrol, but also more toxic compounds against fungi such as pterostilbene and viniferins, were identified and mapped. Their spatial distributions on grapevine leaves irradiated by UV show their specific colocation around the veins. Moreover, MALDI MSI reveals that resveratrol (and piceids) and viniferins are not specifically located on the same area when leaves are infected by Plasmopara viticola. Results obtained by MALDI mass spectrometry imaging demonstrate that this technique would be essential to improve the level of knowledge concerning the role of the stilbene phytoalexins involved in a stress event.