In-vivo quantification of cardiac perfusion is of great research and clinical value. The dual-bolus strategy is universally used in clinical protocols but has known limitations. The dual-saturation acquisition strategy has been proposed as a more accurate alternative, but has not been validated across the wide range of perfusion rates encountered clinically. Dual-saturation acquisition also lacks a clinically-applicable procedure for optimizing parameter selection. Here we present a comprehensive validation study of dual-saturation strategy in vitro and in vivo. The impact of saturation time and profile ordering in acquisitions was systematically analyzed in a phantom consisting of 15 tubes containing different concentrations of contrast agent. In-vivo experiments in healthy pigs were conducted to evaluate the effect of R2* on the definition of the arterial input function (AIF) and to evaluate the relationship between R2* and R1 variations during first-pass of the contrast agent. Quantification by dual-saturation perfusion was compared with the reference-standard dual-bolus strategy in 11 pigs with different grades of myocardial perfusion. Adequate flow estimation by the dual-saturation strategy is achieved with myocardial tissue saturation times around 100 ms (always <30 ms of AIF), with the lowest echo time, and following a signal model for contrast conversion that takes into account the residual R2* effect and profile ordering. There was a good correlation and agreement between myocardial perfusion quantitation by dual-saturation and dual-bolus techniques (R(2) = 0.92, mean difference of 0.1 ml/min/g; myocardial perfusion ranges between 0.18 and 3.93 ml/min/g). The dual-saturation acquisition strategy produces accurate estimates of absolute myocardial perfusion in vivo. The procedure presented here can be applied with minimal interference in standard clinical procedures.