Sepsis is an important cause of morbidity and mortality in neonates and clinicians are typically required to administer empiric antibiotics while waiting for blood culture results. However, prolonged and inappropriate use of antibiotics is associated with various complications and adverse events. Better tools to rapidly rule out bacterial infections are therefore needed. We conducted a prospective observational study aimed to assess the negative predictive value (NPV) of PCR coupled with Electrospray Ionization Mass Spectrometry (PCR/ESI-­‐MS) compared to conventional blood cultures in neonatal sepsis. We found that, in neonates with suspected sepsis, PCR/ESI-­‐MS had a very good NPV of 98% (95% CI : 92%-­‐100%) when compared to blood cultures as gold standard. This novel test might allow for early reassessment of the need for antibiotics.