Infective endocarditis is diagnosed using the Duke criteria, which rely predominantly on cardiac imaging and recovery of a causative organism from the bloodstream. These criteria can be inconclusive, particularly when blood cultures remain sterile either due to the fastidious nature of the infecting organism or prior antibiotic therapy. Serology and, more recently, molecular techniques have been investigated as a solution to the problematic negative blood culture. The detection of elevated antibody levels has proved particularly useful in the diagnosis of those patients infected with organisms that cannot be cultured using standard laboratory methods, whilst molecular methods have been successfully used in the detection of both fastidious pathogens and those inhibited by prior antibiotic therapy. In view of recent and ongoing developments in the field of molecular diagnostics, these techniques will become increasingly important not only in the routine investigation of infectious disease, but specifically the diagnosis of endocarditis.