Current methodologies to measure apoptotic and necrotic cell death using flow cytometry do not adequately differentiate between the two. Here, we describe a flow cytometry methodology adapted to airway epithelial cells (AEC) to sufficiently differentiate apoptotic and necrotic AEC. Specifically, cell lines and primary AEC (n = 12) were permeabilized or infected with rhinovirus 1b (RV1b) over 48 h. Cell death was then measured via annexin V/propidium iodide (A5/PI) or annexin V/TO-PRO-3 (A5/TP3) staining using a novel flow cytometry and gating methodology adapted to AEC. We show that A5/PI staining could not sufficiently differentiate between types of cell death following RV1b infection of primary AEC. However, A5/TP3 staining was able to distinguish six cell death populations (viable, necrotic, debris, A5(+) apoptotic, A5(-) apoptotic, apoptotic bodies) after permeabilization or infection with RV1b, with phenotypic differences were observed in apoptotic populations. Collectively, using a staining and gating strategy never adapted to AEC, A5/TP3 could accurately differentiate and quantify viable, necrotic, and apoptotic AEC following RV1b infection.