The present study investigated the bioactivity of protein hydrolysates and fractionated hydrolysates prepared from brewers' spent grain (BSG) using proteases, including Alcalase 2.4L, Flavourzyme and Corolase PP. Hydrolysates were designated K-Y, including fractionated hydrolysates with molecular weight (m.w.) < 3, <5 and >5 kDa. Where computable, IC50 values were lower in U937 (1.38-9.78%) than Jurkat T cells (1.15-13.82%). Hydrolysates L, Q and R and fractionated hydrolysates of U and W (<3, <5, >5 kDa) significantly (P < 0.01) protected against hydrogen peroxide (H2O2)-induced reduction of superoxide dismutase (SOD) activity. A fractionated hydrolysate of W (<5 kDa) protected against H2O2-induced DNA damage, P < 0.01. Hydrolysates K, N, P, U, U > 5 kDa, V, V > 5 kDa, W, W > 5 kDa significantly (P < 0.05) reduced a concanavlin-A (con-A) stimulated production of interferon-gamma (IFN-gamma). In conclusion, BSG protein hydrolysates demonstrate bioactivity in vitro; lower m.w. hydrolysates (<3, <5 kDa) show greatest antioxidant activity and unfractionated or higher m.w. hydrolysates (>5 kDa) possess anti-inflammatory effects.