We developed a new method for absolute quantification of targeted radiotracers uptake in the myocardium using hybrid SPECT/CT and an external reference point source. A segmentation algorithm based on the level set was developed to determine the endocardial edges from CT, which were subsequently applied to the physically co-registered SPECT. A 3-D Gaussian fitting method was applied for quantification of the external point source. The total targeted radiotracer activity in the myocardium was normalized to that in the point source to calculate the absolute uptake of targeted radiotracer in the myocardium. Preliminary validation was performed in rats with ischemia-induced angiogenesis. The quantified in vivo radiotracer uptake was compared to the postmortem tissue radioactive well-counting of the myocardium. Our methods worked well for identification of the endocardial edges. Quantification of the focal uptake was consistent with the well-counting data. Our methods may have the potential of providing precise absolute quantification of targeted radiotracer uptake in the myocardium.