Abstract. The response of plants to microbial pathogens is based on the production of secondary metabolites. The complexity of plant-pathogen interactions makes their understanding a challenging task for metabolomic studies requiring powerful analytical approaches. In this paper, the ability of ambient mass spectrometry to provide a snapshot of plant metabolic response to pathogen invasion was tested. The fluctuations of glycoalkaloids present in sprouted potatoes infected by the phytopathogen Pythium ultimum were monitored by imprint imaging desorption electrospray ionization mass spectrometry (DESI-MS). After 8 d from the inoculation, a decrease of the relative abundance of potato glycoalkaloids α-solanine (m/z 706) and α-chaconine (m/z 722) was observed, whereas the relative intensity of solanidine (m/z 398), solasodenone (m/z 412), solanaviol (m/z 430), solasodiene (m/z 396), solaspiralidine (m/z 428), γ-solanine/γ-chaconine (m/z 560) , β-solanine (m/z 706), and β-chaconine (m/z 722) increased. The progression of the disease, expressed by the development of brown necrotic lesions on the potato, led to the further decrease of all the glycoalkaloid metabolites. Therefore, the applicability of imprint imaging DESI-MS in studying the plant metabolic changes in a simple pathosystem was demonstrated with minimal sample preparation.