The evaluation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) production by neutrophils is currently a matter of extensive research, with scientific reports showing an enormous variability on the detection methods as well as the concentration of the detecting probes. Also the incubation media used to test neutrophils and the respective ionic concentration, as well as the glucose concentration, varies enormously from study to study. This variability often results in different sensibility and/or response of neutrophils to stimulating agents, which can be a focus of confounding and sometime contradictory results among reports. Thus, the main objective of the present study was to appraise and compare the effect of commonly described buffering media [phosphate buffer saline (PBS), Hank's balanced salt solution (HBSS) and Tris buffer], with or without glucose, on the activation of human neutrophils by phorbol myristate acetate (PMA), using different detection probes [luminol amplified chemiluminescence, dihydrorhodamine 123 fluorescence or cytochrome c reduction (UV/vis spectrometry)]. It was observed that the choice of incubation media as well as the methodology used to detect neutrophils oxidative burst has an enormous influence on posterior results. Independently of buffer, the presence of glucose is important, as a source of NADPH through pentose phosphate pathway (PPP). From the obtained results, we advise the use of HBSS, with the glucose concentration of 0.55mM. This incubation media provided the best performance of neutrophils allowing the use of lower concentrations of the tested probes as well as of the stimulating agent, PMA.