Late-onset neonatal sepsis is one of the leading causes of morbidity and mortality in neonates. Late-onset sepsis is usually defined as sepsis >72 hours after birth. Low birth weight and gestational age are inversely related to the development of late-onset sepsis. Gram-positive organisms appear to be the cause of the majority of the infections. Particularly coagulase-negative staphylococci have been implicated in late-onset neonatal sepsis. The pathophysiological mechanisms underlying the development of sepsis is a complex interaction between the invading pathogen and the neonate’s immune system. Diagnosing late-onset sepsis is extremely challenging due to unspecific symptoms and clinical signs which frequently can mimic other non-infectious etiologies. Blood cultures are time consuming and often yield false negative results. Markers and laboratory test used in the evaluation, such as white blood cell count, absolute neutrophil counts, immature-to-total neutrophil ratio, C-reactive protein, procalcitonin, tumor necrosis factor-α, serum amyloid A, IL-6, IL-8 and CD64 have been used to assist in the identification of late-onset sepsis. However, at present time no marker is individually sufficient to confirm the diagnosis. Usage of two or more markers with different properties, also measured in different time intervals often leads to increased diagnostic accuracy. This could be helpful for the clinicians in the diagnosis and management of late-onset sepsis. There are several novel biomarkers under investigation that appear promising for the future.