Cattle are routinely subjected to painful surgical husbandry procedures, such as castration and dehorning. Quantifying the effectiveness of pain relief interventions during these procedures is challenging, due to the subjectivity and complexity of pain perception in animals and the inherent tendency for prey species to suppress their behavioural responses. Therefore, there are obvious limitations in the use of behavioural observations and routine biochemical or immunological assays restricted to individual targets (e.g. plasma cortisol) for detecting and quantifying the response to pain and stress. One approach, increasingly used in human biomedicine, is to develop an array of plasma biomarkers, which collectively respond to a stimulus. Next generation mass spectrometry techniques, such as SWATH-MS, can be applied to quantitative profiling of proteins (proteomics), lipids (lipidomics) and metabolites (metabolomics) in an unbiased manner and enable simultaneous evaluation of hundreds to thousands of various markers in virtually unlimited number of samples and thus provide more holistic representation of the physiological change. SWATH-MS analysis requires one off construction of spectral libraries which can be expanded as the project develops and shared between laboratories. In this study, SWATH-MS approach has been applied to monitor proteins that form a part of the systemic response to pain and inflammation and are putative targets of analgesic drugs. We describe the development of the foundational data and tools that do not only permit more thorough understanding of pain and inflammation in cattle but also have a potential to advance veterinary medicine in a manner that has not been possible before.