Lupine is a highly sustainable protein source in the Mediterranean region due to its temperature, water, and soil requirements. This work evaluated eight cultivars from different cultivated lupin species —L. albus (Estoril, Celina, and Frieda), L. angustifolius (Giribita and Carabor), and L. luteus (Acos, Cardiga, and a commercial mixture)— in Europe as alternative protein sources. Each cultivar was assessed in terms of proximate composition, phytochemical content, and protein extraction yield. The findings revealed significant inter- and intra-species variability in protein, total dietary fibre, and fat content among the studied cultivars. Lupin protein profile was found to be particularly rich in essential amino acids, including leucine and lysine, as well as beneficial unsaturated fatty acids across all evaluated cultivars. The studied phytochemicals, especially quinolizidine alkaloids, vary significantly among species, while cultivars within the same species showed a more consistent profile. The wet extraction process yielded high-purity protein isolates with favourable extraction efficiency. These findings highlight the potential of lupin for diverse applications in the food and industrial sectors. Cultivar selection is essential to enhance protein extraction yields and colour, as well as to consider lupin as a source of various phytochemicals for valorisation.