Polymorphonuclear neutrophils (PMNs) have multiple functions during the resolution of inflammation. At the inflammation site, increases in chemokines induce infiltration of PMNs via CXCR receptor activation, resulting in cell clearance via phagocytosis and NETosis. Here we describe characterization of the activation and function of PMNs using IncuCyte® live-cell analysis. Changes in cell shape and CD marker expression are known indicators of PMN activation. Freshly isolated PMNs were seeded in 96-well plates, in the presence of FabFlour-488 labeled CD11b antibody for live-cell immunocytochemistry. Phase and fluorescence images were captured every 30 min for 6 h with IncuCyte S3. Cell-by-cell analysis enables individual cell segmentation and yields area, shape (eccentricity) and fluorescent intensity metrics. Subsets of PMNs could be analyzed by classifying on each of these parameters. CXCL8 induced a time- and concentration-dependent increase in eccentricity (EC50 0.63 nM) and concomitant increase in CD11b expression (EC50 0.22 nM). CXCL8-induced activation was attenuated by an anti-CXCL8 antibody. CXCL1 or CCL2 yielded little or no change in either parameter. As a follow up, we assessed CXCL8-dependent chemotaxis using IncuCyte ClearView Chemotaxis plates, phagocytotic activity with IncuCyte pHrodo® E. coli Bioparticles® and PMA-induced NETosis (IncuCyte Cytotox Green). In all three cases robust, time-dependent signal changes were observed, consistent with known PMN function. We conclude that live-cell analysis is a flexible and powerful method for analyzing neutrophil activity, where morphological, protein and functional parameters can be readily quantified and integrated over time.