Brewer's spent grain (BSG) is a valuable byproduct of the brewing industry. It is rich in fibre and proteins, hence an excellent source for fermentative production of bioactive ingredients. This study investigated the development of amino acids and peptides derived from BSG of wheat and stout beers under 10-day fermentation using Lactiplantibacillus plantarum. An in-depth analysis of BSG proteins was carried out using liquid chromatography tandem mass spectrometry. In addition, non-tryptic peptides generated from the BSG fermentation process were identified using data-dependent acquisition mass spectrometry. The identified peptides were screened against a custom-made library to identify potential bioactive peptides and coeliac disease allergens. Bioactive peptides with intense signals were selected for quantification using multiple reaction monitoring mass spectrometry. A total of 503 and 347 proteins were identified in wheat and stour beer BSG, respectively, in which wheat protein sequences were dominant. Fermented wheat beer’s BSG contained a higher number of peptides compared to fermented stout beer’s BSG, with 1,227 versus 436 peptide sequences identified, respectively. Specifically, fermented wheat grains exhibited an increase in peptide numbers in the middle of the fermentation period (day 4), whereas fermented stout grains showed an increase in peptide numbers at the later stage of fermentation (days 8 and 9). Most peptides identified in fermented BSGs were linked to predicted antihypertensive, antimicrobial, and antibacterial properties, while others, derived from wheat, contained intact celiac disease epitopes. Peptide quantitation showed antimicrobial peptides peaking mid-fermentation in both BSG types, with antihypertensive peptides detected early in wheat compared to at the mid-stage for stout. Moreover, free amino acids derived from the BSG’s fermentation were also quantified. After 10-day fermentation, L-alanine, L-leucine, L-proline, and L-glutamic acid increased in both BSG types. Notably, in fermented stout grains, free hydrophobic amino acids (0.98 mg/100g BSG protein) were 60% higher than hydrophilic amino acids (0.61 mg/100g BSG protein), indicating the enzymes in Lb. plantarum could favour the release of hydrophobic amino acids. This study highlights the potential application of fermentation in producing bioactive compounds such as amino acids and peptides from a food by-product.