The aim of this study was to investigate how fractionation of protein and lignin from brewer's spent grain can be influenced by thermochemical and enzymatic pretreatments, including steam explosion, carbohydrase treatment and protease treatment. Following these pretreatments, protein and lignin were partially fractionated by alkaline extraction and acidic precipitation. Steam explosion and hydrolysis of cell wall polysaccharides substantially increased lignin solubilization and its recovery by acidic precipitation. Meanwhile, effective protein extraction required the use of protease, which increased protein solubilization from 15% to nearly 100% from otherwise untreated BSG. Steam explosion reduced the enzymatic protein solubilization but enhanced the extract recovery in the centrifugation step, presumably due to lower water-binding capacity of steam-exploded BSG compared to untreated BSG. Lignin and protein showed clear co-extraction and could only be partially separated by acidic precipitation, suggesting similar solubilities and strong interactions between these components. According to the results, protein and lignin are extractable from BSG after suitable pretreatments. This work provided increased understanding of the chemistry and interactions underlying the challenges in fractionation of BSG components.