Chronic exposure to cigarette smoke can lead to endothelial dysfunction and potentially endothelial cell death, which is often associated with atherosclerosis. Here, we used Human Aortic Endothelial Cells (HAECs) from four healthy donors to determine smoke-induced cytotoxicity. HAECs were exposed to whole smoke conditioned media (WSCM) generated from 3R4F reference cigarettes over a range of 0-8000 ng/mL nicotine equivalence (n.e.) concentrations. Cytotoxicity was evaluated 24 hours post-exposure via neutral red uptake (NRU) and/or reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan for each exposure concentration and compared to vehicle control. The IC50 was then calculated from the concentration-response. Similar IC50 values were observed in both the NRU and MTT assays for the four donors across the dose range of 0-8000 ng/mL n.e. of WSCM. To examine the mechanism responsible for WSCM-induced cytotoxicity in HAECs at varying n.e. concentrations, necrosis (propidium iodide: PI) and apoptosis (Annexin V) markers were assessed via flow cytometry. Annexin V-positive cell populations increased in a dose-dependent manner while increases in PI-positive cell populations only occurred at the highest doses of WSCM (5000-8000 ng/mL n.e.). To further investigate the induction of apoptotic cells after WSCM exposure, we conducted Western blotting experimentation using caspase-3 cleavage as a marker for apoptosis. Western blots confirmed that apoptosis occurs at the higher concentrations of WSCM which coincide with reduced HAEC survival. We conclude that WSCM produces comparable cytotoxicity in vitro when applied to HAECs from four donors as assessed using both the NRU and MTT assays. Additionally, the mechanism of toxicity appears to be dose-dependent and conserved among the four donors as assessed by distinct measures for apoptosis.