Leaf rust resistance is of high importance for a sustainable European wheat production. The expression of known resistance genes starts at different developmental stages of wheat. Breeding for resistance can be supported by a fast, precise, and resource-saving phenotyping. The examination of detached leaf assays of juvenile plants inoculated under controlled conditions and phenotyped by a robotic- and computer-based, high-throughput system is a promising approach in this respect. Within this study, the validation of the phenotyping workflow was conducted based on a winter wheat set derived from Central Europe and examined at different plant developmental stages. Moderate Pearson correlations of 0.38–0.45 comparing leaf rust resistance of juvenile and adult plants were calculated and may be mainly due to different environmental conditions. Specially, the infection under controlled conditions was limited by the application of a single rust race at only one time point. Our results suggest that the diversification with respect to the applied rust race spectrum is promising to increase the consistency of detached leaf assays and the transferability of its results to the field.