Background: Lupin is a promising legume crop, belongs to the Fabaceae (or Leguminosae) family. Lupin production for traditional and functional foods or animal feed is limited, due to the content of toxic quinolizidine (QA)s and indole alkaloids (IA)s. These compounds may not only pose a risk to humans and animals through food consumption, but may also affect soil and aquatic ecosystems. Field experiments were conducted to study the alkaloids content in both narrow-leaved or blue (L. angustifolius) and yellow (L. luteus) lupin plant tissue during a full growing season and understand the environmental fate of alkaloids in soil and water. Suction cups were used to collect soil pore water (soil solution) at four depths: 10, 25, 50 and 70 cm. A full protocol for sample preparation and UPLC–MS/MS quantification of alkaloids in plant, soil and water was developed. Results: During the field experiments the alkaloids in the plant tissues increased, at the harvest stage the content was highest with 21.4 and 24.6 mg/kg dry weight (dw) for blue and yellow lupin, respectively. In soil, alkaloids quantified during the growing season (max concentration was 1.3 × 102 µg/kg dw) and even detected after harvest (0.2 µg/ kg dw). In soil pore water samples, alkaloids were not detected during summer, but the concentrations increased to 9.8 × 102 and 1.5 × 103 ng/L for blue and yellow, respectively, in September when autumn precipitation began. Conclusions: The results show the amount of alkaloids transferred from plant tissue into soil and soil pore water estimated to be on average 0.016% and 0.005% in soil and soil pore water, respectively. Alkaloids leached from topsoil to subsoil layers; the concentrations decline with soil depth. This study demonstrates that alkaloids are mobile compounds in the soil environments, thus lupin production may affect soil or aquatic ecosystems, and reduce water quality.