The current research on the epidemiology and diagnosis of Brucella, also stimulated by the zoonotic and bioterrorism aspect, is exploiting novel methods to develop more rapid, reliable and safe diagnostic tools. This paper evaluates the various diagnostic strategies to suggest the most reliable technique to diagnose Brucella spp. infection in bovines. The pros and cons of phenotypic identification, serological diagnosis as well as genotypic identification have been discussed. MALDI-TOF-MS technology has been found to have significant advantages over other conventional and molecular identification methods and can be used as a first-line screening tool for epidemiological studies and outbreak investigations of bovine brucellosis with minimal time, labor and cost.