Brewers’ spent grain (BSG), the main by-product of the brewing industry, is a rich source of minerals and water-soluble vitamins such as thiamine, pyridoxine, niacin, and cobalamin. Bioaccessibility through in vitro digestion is an important step toward the complete absorption of minerals and B group vitamins in the gastrointestinal system. Inductively coupled plasma optical emission spectrometry (ICP-OES) together with inductively coupled plasma quadrupole mass spectrometry (ICP-MS) was used for the quantification of the macro- and micro-minerals. An ultra-high performance liquid chromatography (UHPLC) system coupled with a diode array detector (DAD) was used for B group vitamin identification. Four different industrial BSG samples were used in the present study, with different percentages of malted cereals such as barley, wheat, and degermed corn. Calcium’s bioaccessibility was higher in the BSG4 sample composed of 50% malted barley and 50% malted wheat (16.03%), while iron presented the highest bioaccessibility value in the BSG2 sample (30.03%) composed of 65% Pale Ale malt and 35% Vienna malt. On the other hand, vitamin B1 had the highest bioaccessibility value (72.45%) in the BSG3 sample, whilst B6 registered the lowest bioaccessibility value (16.47%) in the BSG2 sample. Therefore, measuring the bioaccessibilty of bioactive BSG compounds before their further use is crucial in assessing their bioavailability.