Purpose: To develop a novel in vitro method of evaluating coronary artery ischemia using a combination of noninvasive coronary CT angiograms (CCTA) and 3D printing. Materials and Methods: Five patients with varying degrees of coronary artery disease who underwent non-invasive CCTA scans and invasive fractional flow reserve (FFR) of their left anterior descending coronary artery (LAD) were included in this study. The LAD artery was segmented and reconstructed using Mimics (Materialise). The segmented models were then 3D printed using a Carbon (Carbon Inc.,) and Objet260 Connex (Stratasys) printers with urethane methacrylate (UMA) family of rigid resins and Veroclear, respectively. An in vitro flow circulation system representative of invasive measurements in a cardiac catheterization laboratory was developed to experimentally evaluate the hemodynamic parameters of pressure and flow. Physiological coronary circulation was modelled in vitro as flow‐dependent stenosis resistance in series with variable downstream resistance. A range of physiological flow rates was applied by a peristaltic steady flow pump and titrated by a flow sensor. The pressure drop and the pressure ratio (Pd/Pa) were assessed for patient-specific aortic pressure and differing flow rates to evaluate FFR in vitro. Results: For these five models, there was a good positive correlation (r = 0.78) between the in vitro and invasive FFR. The mean differences, as assessed by Bland-Altman analysis, between in vitro and invasively measured FFR was at 0.01±0.1 (95% limit of agreement -0.1169 to 0.1369). Conclusions: 3D printed patient-specific models can be used in a non-invasive in vitro environment to quantify coronary artery ischemia as assessed by invasive FFR.