Anthracnose, caused by Colletotrichum acutatum, is the most destructive fungal disease of Andean lupin (Lupinus mutabilis Sweet) in Ecuador and of other lupin species around the world. Symptoms of necrotic spots occur throughout the main stem, and infection progresses to cause bending of the main stem and lateral branches, resulting in yield loss. Although there is no known anthracnose resistance, this study aims to assess tolerance of Andean lupin and investigate lupin–C. acutatum interactions. Two Andean lupin genotypes, I-450 Andino and I-451 Guaranguito, were inoculated on the meristematic section of the main stem, either by spraying or by pipetting C. acutatum spores on to an artificial wound. Although the two methods gave similar results, spraying is the preferred method because it mimics natural pathogen infection. Plant-pathogen interactions were assessed at five different phenological stages (leaf stages 2–3, 4–5, 6–7, 8–9, and 10–11) with three C. acutatum isolates by using a 0–5 scale to assess disease symptoms. In both genotypes, anthracnose symptoms were greater at early seedling stage (2–3-leaf stage), decreasing significantly in early vegetative phase (6–7-leaf stage) and increasing again when the flower stage began (10–11-leaf stage). However, the tolerance of these two Andean lupin genotypes to anthracnose was not equally expressed at all developmental stages. We recommend, in a breeding program, that screening for anthracnose first occurs at the 6–7-leaf stage (6 weeks old) and again when flowering starts at the 10–11-leaf stage (10 weeks old) so that the overall tolerance can be determined. This method could be used in lupin breeding programs for improving resistance to anthracnose.