Neutrophil extracellular trap (NET) formation is a recently discovered process in the field of innate immunity. It is important to have consistent standards in inducing and quantifying NET formation to compare data from different labs in this new area of investigation. Here we describe the conditions of neutrophil isolation from peripheral blood and stimulation that we find allow the study of NETosis in vitro. The criteria for conclusively identifying the process of NETosis, and the pros and cons of various quantification methods are discussed.