The only definitive method for successfully identifying the vaccinated animals in the presence of an active infection is to determine the rate of pathogen shedding by isolation, cytokine/interleukin profiling or neutralization assay. These types of analysis require repeated sampling, a period for seroconversion and expensive, therefore not routinely employed during endemic infection outbreaks. Differentiating infected from vaccinated animals (DIVA) marker vaccines (e.g. a modified wild type virus with a gene deletion resulting in the absence of a particular diagnostic antigen) can be employed to differentiate vaccine antibody responses from that of wild type pathogen. Companion serology based tests rely on seroconversion, and upon exposure to wild type pathogen, the antibody response to DIVA vaccines will be masked. A potential approach that can meet this limitation is based on the application of metabolomics to identify metabolites or 'small molecules' in biological samples that are signatures that correlate or provide some evidence of immune protection. These metabolites are often the end stage products of biological processes and therefore provide an accurate representation of an organism's homeostatic status at the time of sampling. Metabolomic analysis of bio-fluids has provided new insights to the understanding of the patho-physiological processes involved in disease establishment; development and diagnosis. The application of metabolomic profiling may help to overcome the current limitations in DIVA diagnostics by identifying markers capable of differentiating between vaccinated and non-vaccinated animals, and importantly allow the development of better tools to assess the performance of vaccines.