Brewer's spent grain (BSG) is a by-product of the beer industry and a potential source of bioactive compounds. In this study, two methods of extracting bioactive compounds from brewer's spent grain were used - solid-to-liquid conventional extraction (SLE) and solid-to-liquid ohmic heating extraction (OHE) coupled with two ratio combinations of solvents: 60 % and 80 % ethanol:water (v/v). The bioactive potential of the BSG extracts was assessed during the gastrointestinal tract digestion (GID) and the differences in their antioxidant activity, total phenolic content and characterization of the polyphenol profile was measured. The SLE extraction using 60 % ethanol:water (v/v) was the extraction method with higher antioxidant activity (33.88 mg ascorbic acid/g BSG - initial; 16.61 mg ascorbic acid/g BSG - mouth; 15.58 mg ascorbic acid/g BSG - stomach; 17.26 mg ascorbic acid/g BSG - duodenum) and higher content in total phenolics (13.26 mg gallic acid/g BSG - initial; 4.80 mg gallic acid/g BSG - mouth; 4.88 mg gallic acid/g BSG - stomach; 5.00 mg gallic acid/g BSG - duodenum). However, the OHE extraction using 80 % ethanol:water (v/v), had a higher bioaccessibility index (99.77 % for ferulic acid, 72.68 % for 4-hydroxybenzoic acid, 65.37 % for vanillin, 28.99 % for p-coumaric, 22.54 % for catechin) values of polyphenols. All the extracts enhanced (except for SLE for 60 % ethanol:water (v/v) at 2 and 1.5 %, and for 80 % ethanol:water (v/v) at 2 % with Bifidobacterium animalis spp. lactis BB12, where no growth was observed) the growth of the probiotic microorganisms tested (Bifidobacterium animalis B0 - O.D.'s between 0.8240 and 1.7727; Bifidobacterium animalis spp. lactis BB12 - O.D.'s between 0.7219 and 0.8798; Lacticaseibacillus casei 01 - O.D.'s between 0.9121 and 1.0249; and Lactobacillus acidophilus LA-5 - O.D.'s between 0.8595 and 0.9677), demonstrating a potential prebiotic activity of BSG extracts.