Barley ( Hordeum vulgare ) is the fourth most cultivated crop in the world in terms of production volume, and it is also the most important raw material of the malting and brewing industries. Barley belongs to the grass (Poaceae) family and plays an important role in food security and food safety for both humans and livestock. With the global population set to reach 9.7 billion by 2050, but with less available and/or suitable land for agriculture, the use of biotechnology tools in breeding programs are of considerable importance in the quest to meet the growing food gap. Proteomics as a member of the “omics” technologies has become popular for the investigation of proteins in cereal crops and particularly barley and its related products such as malt and beer. This technology has been applied to study how proteins in barley respond to adverse environmental conditions including abiotic and/or biotic stresses, how they are impacted during food processing including malting and brewing, and the presence of proteins implicated in celiac disease. Moreover, proteomics can be used in the future to inform breeding programs that aim to enhance the nutritional value and broaden the application of this crop in new food and beverage products. Mass spectrometry analysis is a valuable tool that, along with genomics and transcriptomics, can inform plant breeding strategies that aim to produce superior barley varieties. In this review, recent studies employing both qualitative and quantitative mass spectrometry approaches are explored with a focus on their application in cultivation, manufacturing, processing, quality, and the safety of barley and its related products.