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Soybean (Glycine max) is one of the most important crops in the world. Phakopsora pachyrhizi is a plant pathogenic basidiomycete fungus that infects soybean, causing Asian Soybean Rust (ASR) disease and affecting production. Here, we describe how to prepare the plant material and the uredospore suspension (from spores harvested from leaves exhibiting sporulating uredinia) for in vitro leaf infection. Plant material is sprayed with the uredospore suspension and incubated for 12 days. During the incubation period, the presence of lesions and pustules is visually verified. After this incubation period, the leaves are classified according to the lesion type. The number of uredospores per CM2 of leaf was also estimated. The detached-leaf assay is routinely used to test fungicide efficiency (Scherb and Mehl, 2006). Detached-leaf, greenhouse and field results have been shown to be significantly correlated (Twizeyimana et al., 2007). The present protocol was adapted from the two publications cited above. The usefulness of this approach for studying P. pachyrhyzi infection on transgenic soybean was previously demonstrated by our research team (Wiebke-Strohm et al., 2012; Bencke-Malato et al., 2014).