Reactive oxygen species (ROS) production by phagocytic leukocytes is a critical factor in immunity against microorganisms. Human neonates are susceptible to overwhelming infections, for which abnormal granulocyte function may play an important role. In this study, we aimed to identify a convenient and quantitative method to measure ROS production by human granulocytes after cellular activation. We first compared the results of a flow cytometric assay with 2',7'-dichlorodihydrofluorescein diacetate (H2DCF) probe or dihydrorhodamine-123 (DHR123) and an enhanced chemiluminescence test using granulocytes from a patient with chronic granulomatous disease and normal granulocytes stimulated with different concentrations of phorbol myristate-13-acetate. Peripheral blood granulocyte respiratory burst ability from 37 newborn babies with different gestational ages was then quantified using flow cytometric assay with H2DCF probe. We found that flow cytometric assay of ROS production is sensitive and correlates with chemiluminescence measurements. The results showed that activated granulocytes from neonates with higher birth body weights and more advanced gestational ages tend to have higher levels of ROS production in respiratory burst (p=0.0042 and 0.063, respectively). This study demonstrates that flow cytometry is suitable for detecting the functional defects in granulocyte ROS production in human neonates of different gestational age.