A rapid and simple method for measurement of respiratory burst in neutrophil granulocytes in whole bovine blood is described. The respiratory burst was stimulated by live Staphylococcus aureus, and the production of reactive oxygen species quantified by the conversion of intracellular dihydrorhodamine 123 to the green fluorescent rhodamine 123, measured by flow cytometry. Assay conditions, including bacterial and dihydrorhodamine 123 concentrations and incubation time, were determined. Repeatability and precision of the method were assessed by testing parallel samples from clinically healthy dairy cows. In vitro and in vivo inhibition of respiratory burst was investigated, and labelling with a granulocyte marker antibody was performed. Stimulation with live S. aureus induced green fluorescence in the neutrophil granulocytes in a whole blood preparation. The fluorescence intensity increased with increasing bacterial concentration and increasing incubation time. Agreement analysis showed that the method gave repeatable results, and the intra-assay variability of the method was relatively low. The method is considered a useful technique for measurement of neutrophil respiratory burst in whole bovine blood.