The aim of the present study was to design an automated-gating hematology fluorescence flow cytom-etry methodology permitting the assessment of neutrophil and monocyte activation in EDTA-anticoa-gulated whole blood based on cell granularity, lipid membrane components, cell shape and volume, and total cell nucleic acid (NA) compounds. For particularly monitoring the proper functioning of patients' innate immune system as the first line defense against microbial invaders, the suitable test system should be rapid, simple, reliable by yielding reproducible results. It must be validated against established methods, and it must prove to work in selected clinical settings, e.g. in intensive care unit (ICU) environments. The adaptation of a routine hematology cell analyser utilizing fluorescence flow cytometry resulted in a potentially useful system for all requirements. It proved to detect in real-time and in a reliable and reproducible way the main cellular response reactions of neutrophils and monocytes during externally stimulated immune defense. Validation was successful when comparing it to established methods. The quantified activation effects were dose dependent from the applied activating agents. Cellular response kinetics could be measured and described and showed to be in line with the prevailing cell response models. Upon applying the test method to a healthy population of volunteers and a first cohort of ICU patients with and without evident immune depression, the test revealed excellent cellular responses to external activating cytotoxic stimuli (lipopolysaccharide; LPS) for the control group, slightly weaker response from ICU patients without immune depression and no response from patients with evident immune depression. We conclude that routine hematology fluorescence flow cytometry can accurately and reproducibly measure different activation steps of monocytes and polymorphonuclear neutrophilic granulocytes to defined external stimuli. This may potentially be applied as a STAT (Latin statim 5 immediately) and routine screening and surveillance method for inflammatory diseases. q 2008 Clinical Cytometry Society Key terms: automated rapid method; innate immune activation; ex vivo LPS or fMLP stimulation; Sysmex XE2100; ICU patients How to cite this article: Linssen J, Aderhold S, Nierhaus A, Frings D, Kaltschmidt C, Zänker K. Automation and validation of a rapid method to assess neutrophil and monocyte activation by routine fluorescence flow cytome-try in vitro. Cytometry Part B 2008; 74B: 295-309. The human innate immune system is of vital importance to defend the organism against the ubiquitous microbial enemies. Besides its humoral part, the complement system, core component is the cellular immune system comprising monocytes (M) and polymorphonu-clear neutrophilic granulocytes (PMN) in peripheral