The high incidence of neonatal sepsis worldwide and the considerably high mortality rate of severe sepsis and septic shock call for an earlier diagnosis and more accurate monitoring of the disease. Conventional laboratory tests, such as white blood cell count (WBC) and differential count, micro-erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) have a number of limitations associated with their limited sensitivity in the early phase of the disease and their non-specific increase in the course of various severe neonatal clinical conditions like asphyxia, meconium aspiration and prolonged rupture of membranes. Next-generation biomarkers encompass new molecular tests, accurate measurement of the proteins and enzymes mainly involved in the innate immunity biochemical pathways, application of proteomics and metabolomics for risk stratification and prognosis, and the clinical use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of various bacteria and yeasts. The availability of sophisticated biochemical and molecular tests and of innovative technologies can significantly improve baby outcomes in terms of earlier and more accurate diagnosis, tailored therapeutic treatment, shorter hospitalization and thus minimized complications, and ultimately can prevent and monitor nosocomial and healthcare-associated infections. As a consequence, costs can be significantly reduced over a full cycle of care by investing in high quality laboratory medicine.