Progress has been made in the reduction of morbidity and mortality from neonatal sepsis. However, diagnosis continues to rely primarily on conventional microbiologic techniques, which can be inaccurate. The objective of this review is to provide the clinician with an overview of the current information available on diagnosing this condition. We review currently available diagnostic approaches for documenting neonatal sepsis and also describe novel approaches for diagnosing infection in neonates who are under development and investigation. Substantial progress has been made with molecular approaches and further development of non-culture-based methods offer promise. The potential ability to incorporate antimicrobial resistance gene testing in addition to pathogen identification may provide a venue to incorporate a predominantly molecular platform into a larger program of neonatal care.