Infection efficiency is a key epidemiological parameter that determines the proportion of pathogen spores able to infect and cause lesions once they have landed on a susceptible plant tissue. In this study, an improved method to measure infection efficiency of Zymoseptoria tritici using a replicated greenhouse experiment is presented. Zymoseptoria tritici is a fungal pathogen that infects wheat leaves and causes septoria tritici blotch (STB), a major disease of wheat worldwide. A novel experimental setup was devised, where living wheat leaves were attached to metal plates, allowing for time‐resolved imaging of disease progress in planta. Because lesions were continuously appearing, expanding and merging during the period of up to 3 weeks, daily measurements were necessary for accurate counting of lesions. Reference membranes were also used to characterize the density and spatial distribution of spores inoculated onto leaf surfaces. In this way, the relationship between the number of lesions and the number of viable spores deposited on the leaves was captured and an infection efficiency of about 4% was estimated from the slope of this relationship. This study provides a proof of principle for accurate and reliable measurement of infection efficiency of Z. tritici. The method opens opportunities for determining the genetic basis of the component of quantitative resistance that suppresses infection efficiency. This knowledge would improve breeding for quantitative resistance against STB, a control measure considered more durable than deployment of major resistance genes.