Introduction: Tobacco smoke is the single most significant modifiable risk factor of cardiovascular disease (CVD), yet the contribution of electronic cigarettes (e-cig) to CVD risk is uncertain. Objective: The purpose of this study was to test an ex vivo approach for evaluation of vascular toxicity of tobacco products including e-cig aerosols. Methods: The assay relies on 2 facts of aortic function: 1) it is a biomarker of endothelial dysfunction; and, 2) it is nearly 100% dependent on endothelial-derived nitric oxide synthase (eNOS). To assess real-time eNOS function, phenylephrine-precontracted (PE) murine aortas were incubated with L-nitroargininemethylester (L-NAME) that subsequently increased tension and blocked (>99%) acetylcholine-induced relaxation (ACh %). The ‘L-NAME PE Contraction Ratio’ (PECR) was calculated as: ‘PE Tension post-L-NAME’ divided by ‘PE Tension pre-L-NAME’, and regressed vs ACh % relaxation. Results: Validation 1: PECR and ACh % values of naïve aortas were strongly and positively correlated (PECR vs ACh %, r 2 =0.91, n=7). Validation 2: Retrospective analyses of published PECR and ACh % data of aortic function of female mice exposed to air, propylene glycol:vegetable glycerin (PG:VG), formaldehyde (FA), or acetaldehyde (AA) for 4d, 6h/d showed that the PECR in air-exposed mice (PECR=1.42±0.04, n=16) correlated positively with the ACh % (r 2 =0.40) as in naïve aortas. Similarly, PECR values were significantly decreased in aortas with endothelial dysfunction yet retained positive regression coefficients (PG:VG r 2 =0.54; FA r 2 =0.55). Unlike other toxicants, AA exposure significantly increased both PECR and ACh % values yet diminished regression (r 2 =0.09). Validation 3: Naïve aortas were incubated (1h) with particles of either mainstream cigarette smoke (MCS) or PG:VG, and the PECR was significantly increased by MCS (1.97±0.17; p<0.05) yet not by PG:VG (1.34±0.05) compared with controls (1.48±0.05) indicating MCS induced vascular toxicity. Conclusions: Because the PECR reflects eNOS function directly, it is a robust tool for evaluating tobacco toxicity and predicting endothelial dysfunction. Thus, it strongly complements and extends insights gained using ex vivo measures of vascular toxicity.