Neutrophil Extracellular Traps (NETs) are a crucial host defense mechanism employed by neutrophils in response to various aggregates and contribute to pathology in diverse diseases, from chronic inflammation to sepsis. This protocol details the preparation of stimulated differentiated HL-60 cells with Phorbol 12-myristate 13-acetate followed by the process of immunostaining and use of a high-throughput microscope, the Nanolive. The release of NETs, also known as NETosis, is further quantified using readily available equipment, including the CellProfiler, an innovative analyzer.