Background Necrotizing pathogens pose an immense economic and ecological threat to trees and forests, but the molecular analysis of these pathogens is still in its infancy because of lacking model systems. To close this gap, we developed a reliable bioassay for the widespread necrotic pathogen Botrytis cinerea on poplars (Populus sp.), which are established model organisms to study tree molecular biology. Results Botrytis cinerea was isolated from Populus x canescens leaves. We developed an infection system using fungal agar plugs, which are easy to handle. The method does not require costly machinery and results in very high infection success and significant fungal proliferation within four days. We successfully tested the fungal plug infection on 18 poplar species from five different sections. Emerging necroses were phenotypically and anatomically examined in Populus x canescens leaves. We adapted methods for image analyses of necrotic areas. We calibrated B. cinerea DNA against Ct-values obtained by quantitative real-time polymerase chain reaction and measured the amounts of fungal DNA in infected leaves. Increases in necrotic area and fungal DNA were strictly correlated within the first four days after inoculation. Methyl jasmonate pretreatment of poplar leaves decreased the spreading of the infection. Conclusions We provide a simple and rapid protocol to study the effects of a necrotizing pathogen on poplar leaves. The bioassay and fungal DNA quantification for Botrytis cinerea set the stage for in-depth molecular studies of immunity and resistance to a generalist necrotic pathogen in trees.