Coronary collateral circulation is an alternative source of blood supply to the myocardium when the original blood flow is not sufficient. The pressure derived collateral flow index (CFI) calculated with (P w - P v)/(P a - P v) can be measured during angioplasty and predicts the outcome of the patients with ischemic heart disease. However, the necessity of occluding the coronary artery at maximum hyperemia hampers the wide spread use. We mathematically derived a new equation which calculates CFI in sequential stenoses. The present study aimed to validate the equation in an in vitro model of coronary circulation. The predicted CFI is calculated from the following equation: CFI = (FFRpre - FFRpost(1 - ∆FFR))/(FFRpre + ∆FFR - FFRpost). FFRpre/post is the FFR before and after the angioplasty. ∆FFR is the FFR gradient across the target stenosis. The experimental system consisted of a pump, systemic circulation, coronary circulation, and 5 constrictors placed in the coronary artery. A total of 30 different sequential coronary stenoses were randomly created and 30 different pressure data were obtained. The predicted CFI was compared with the true CFI. A close positive correlation (r = 0.90, p < 0.0001) was found between the predicted CFI and the true CFI. The accuracy of the equation was demonstrated in an in vitro model of coronary circulation.