Spring frost poses a challenge for all major crops and, in the case of Lupinus mutabilis (Andean lupin) can cause severe damage or even total loss of the crop. Within the LIBBIO project consortium, we conducted a series of experiments in order to develop a suitable protocol for screening lupin germplasm under frost-simulation conditions. Four lupin accessions, one Lupinus albus and three Andean lupins were used in the experiments (L. albus Mihai, L. mutabilis LIB 220, LIB 221, LIB 222). Seedlings at four developmental stages were challenged with five different levels of \u2018frost\u2019 stress from low (\u22122 \u00b0C) to high (\u221210 \u00b0C). Notably, young seedling (cotyledons just breaking through the soil surface) showed little evidence of frost damage for temperatures down to \u22126 \u00b0C. At \u22128 \u00b0C, however, damage was evident, suggesting a cold tolerance threshold occurs at this temperature. Interestingly, for later developmental stages, when the first and second leaves were visible, notable differences were observed starting at \u22126 \u00b0C. The results indicate that the plant growth stage is an important parameter when screening for frost tolerance in germplasm. Overall, by identifying Andean lupin genotypes adapted to high abiotic stress factors, farmers will be able to use it as a reference crop with potentially a commercial interest from the food sector, or cosmetics, and biofuel industries.