Background: The diagnosis of neonatal sepsis is challenging due to non-specific and subtle clinical features, low sensitivity and delay in routine laboratory tests. Current study was conducted to evaluate the role of manual immature/total (I/T) neutrophil ratio and automated immature granulocyte count (IGC) and immature granulocyte percentage (IG%) in the diagnosis of neonatal sepsis. Materials and Methods: An analytical cross-sectional study was done during a period of 6 months with a sample of 55 neonates admitted to Colombo South Teaching Hospital, Sri Lanka. A combination of clinical and laboratory parameters including full blood count, C-reactive protein and blood culture were used to identify the neonates with probable sepsis. The population was subcategorized into five (5) groups and manual immature/total neutrophil (I/T) ratio, immature granulocyte count (IGC) and immature granulocyte%(IG%) were done in each neonate. Results: The sensitivity of manual I/T ratio was 93.75% and negative predictive value (NPV) was 95.24%. The sensitivity for lower cut off values, IGC of 0.03x103/µL and IG% of 0.5% was 80% and 73.33% respectively. The NPV for above cut-off values were 25% and 0.5% respectively. The NPV was improved with higher cut-off values with 70.90% for IGC 0.3 and 70.59% for IG 3%, but sensitivity remained low with 40% and 33.33% respectively.Conclusion: Manual I/T ratio remains as a useful diagnostic tool in diagnosing and excluding neonatal sepsis with a very good sensitivity and NPV. However, further studies and well defined reference intervals are required in automated IGC and IG%.