Protein-enriched isolates and co-product fractions were obtained from sheared, pale and black brewers' spent grain (BSG) using sequential aqueous and alkaline (110 mm NaOH) extraction, followed by isoelectric precipitation at pH 3.8. A recovery of 59% of the original pale BSG protein and 15% of the black BSG protein was obtained for the final isolates. Gel permeation HPLC (GP-HPLC) revealed that 59% of the extracted pale BSG protein and only 6% of black BSG protein had a molecular mass >10 kDa. Glutamine/glutamate and proline were the most abundant amino acids present in both isolates. Analysis of four co-product fractions obtained during fractionation from both pale and black BSG revealed the presence of phenolics, with higher concentrations in the black BSG extracts. These fractions possessed antioxidant and free radical scavenging activity when tested using the ferric reducing ability of plasma (0.16 ± 0.01 to 4.33 ± 0.11 mg Trolox equivalents g−1 BSG dry weight) and diphenylpicrylhydrazyl (12.85 ± 1.16% to 59.50 ± 3.47% DPPH۠sc) assays, respectively. The protein-enriched isolates and the phenolic-rich extracts may find use as value-added ingredients for incorporation into conventional and functional foods.