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Protein hydrolysates are largely used as plant biostimulants for boosting crop growth, and improving crop tolerance to abiotic stresses and fruit quality. Protein hydrolysate-based biostimulants are mostly produced by chemical hydrolysis starting from animal wastes. However, an innovative process of enzymatic hydrolysis of legume-derived proteins has been recently introduced by few companies. The objective of this study was to evaluate the energy use and environmental impact of the production processes of enzymatically-produced protein hydrolysate starting from lupine seeds and protein hydrolysate obtained from chemical hydrolysis of leather wastes through the application of life cycle assessment (LCA). The LCA method was applied through the software GEMIS “Global Emission Model for Integrated Systems”, elaborated at L’Oko-Institute in Germany, and the parameters taken into account were: CO2 emissions in g per kg of protein hydrolysate; the consumption of fossil energy expressed in MJ per kg of protein hydrolysate; and water consumption reported in kg per kg of protein hydrolysate. In the case of legume-derived protein hydrolysate, the evaluation of the energy use and the environmental impact started from field production of lupine grains and ended with the industrial production of protein hydrolysate. In the case of animal-derived protein hydrolysate, the LCA method was applied only in the industrial production process, because the collagen is considered a waste product of the leather industry. The type of hydrolysis is the step that most affects the energy use and environmental impact on the entire industrial production process. The results obtained in terms of CO2 emissions, fossil energy consumption and water use through the application of LCA showed that the production process of the animal-derived protein hydrolysate was characterized by a higher energy use (+26%) and environmental impact (+57% of CO2 emissions) in comparison with the enzymatic production process of lupine-derived protein hydrolysate. In conclusion, the production of legume-derived protein hydrolysate by enzymatic hydrolysis is more environmentally friendly than the production of animal-derived protein hydrolysate through chemical hydrolysis.