‘One Health’ summarises the idea that human health and animal health are interdependent and bound to the health of ecosystems. The purpose of proteomics methodologies and studies is to determine proteins present in samples of interest and to quantify changes in protein expression during pathological conditions. The objectives of this paper are to review the application of proteomics technologies within the One Health concept and to appraise their role in the elucidation of diseases and situations relevant to One Health. The paper develops in three sections. Proteomics Applications in Zoonotic Infections part discusses proteomics applications in zoonotic infections and explores the use of proteomics for studying pathogenetic pathways, transmission dynamics, diagnostic biomarkers and novel vaccines in prion, viral, bacterial, protozoan and metazoan zoonotic infections. Proteomics Applications in Antibiotic Resistance part discusses proteomics applications in mechanisms of resistance development and discovery of novel treatments for antibiotic resistance. Proteomics Applications in Food Safety part discusses the detection of allergens, exposure of adulteration, identification of pathogens and toxins, study of product traits and characterisation of proteins in food safety. Sensitive analysis of proteins, including low-abundant ones in complex biological samples, will be achieved in the future, thus enabling implementation of targeted proteomics in clinical settings, shedding light on biomarker research and promoting the One Health concept.