Legumes are an important source of protein, lipids, and other essential nutrients. As the demand for protein and lipids continues to surge on a global scale, there is a growing interest in incorporating legumes into aquafeeds. This shift is driven not only by the escalating growth of the aquaculture sector in recent years but also by the imperative to diminish the dependency on traditional resources like fishmeal (FM) and fish oil. Amongst legumes, different lupin species had been identified as a potential protein source to partially reduce the inclusion of FM in countries such as Australia, Chile, and the European Union. A comprehensive evaluation of their nutritional profiles, overall characteristics, and potential antinutritional factors is essential for informed utilization and the implementation of nutritional enhancement strategies. In pursuit of this goal, an in vitro gastrointestinal simulation system was devised to replicate the digestive conditions of rainbow trout (Oncorhynchus mykiss). The study focused on determining the bioaccessibility of protein and phosphorus within two sweet lupin varieties (alkaloids < 0.05) with high (Lupinus mutabilis) and low (Lupinus angustifolius) native phytic acid content evaluated as whole (W) or dehulled (D) seeds meals and the effect of a single dose of phytase (2,500 FTU/kg DM). Additionally, regular soybean meal (SBM) served as reference (10 treatments with 3 replicates). A 2,500 FTU/kg DM phytase dose increased the levels of PO4−3 released throughout the intestinal phase by 122.6% for L. mutatabilis W, 116.3% for L. mutatabilis D, 65.2% for L. angustifolius W, 59.0% for L. angustifolius D, and 91.8% for SBM compared to controls without phytase. The bioaccessibility of amino acids in varieties treated with phytase increased with respect to the control without phytase. L. mutabilis was found to be a potentially viable alternative as a good quality protein source for the manufacture of environmentally friendly aquafeeds.