Tobacco broomrape (Phelipanche ramosa) is a widespread parasitic plant that causes serious damage in some countries. Selection of resistant varieties is an essential pillar of an integrated broomrape control strategy but research performed on broomrape resistance until now is tedious and time consuming because phenotyping, through germination rate measurement, is usually evaluated by counting germinated seeds under a binocular microscope. The objective of this study was to adapt to tobacco a method developed on rapeseed by the team at the French University of Nantes. Their publication describes a fast and easy technique for broomrape germination rate determination based on a standardised 96 well plate test coupled with spectrophotometric reading. Twenty tobacco and related species genotypes with tolerance potential were selected for this study. The two controls, tolerant vs susceptible, were Wika and ITB 31612. Root exudates were sampled from individual plants during six weeks and deposited on broomrape non-dormant seeds. GR24 was used as the positive germination control. After staining treatment with methylthiazolyldiphenyl-tetrazolium bromide (MTT), spectrophotometric reading results were collected. After four weeks of exudates collection, significant average germination rates of 8 % on the reference tolerant genotype and 40 % on the reference of susceptible genotypes were obtained. Initial work on these two control genotypes has been key in defining the best test conditions for tobacco. Evaluation of the 20 genotypes allowed detection of a potential in other resistance sources that are explored more largely in current programmes. A high-throughput phenotyping tool, such as this test, could support our resistance breeding strategy; the higher the resolution of the phenotype analysis, the more likely that new genes and complex interactions will be revealed.