Localized magnetic resonance spectroscopy (LMRS) promises a powerful non-invasive means to determine myocardial triacylglycerol (TAG) in a clinical setting. Here, the linearity, specificity, robustness, precision, and accuracy of an ex vivo mouse-heart LMRS TAG assay are assessed by quantifying the spatial, spectral, and relaxation-induced uncertainties. The protocol, which is based on localization by adiabatic selective refocusing (LASER) using frequency offset corrected inversion (FOCI) pulses, alternating gradient polarity, and simple post-processing, is shown to have good characteristics. The presented protocol has a benchmark, phantom-based, accuracy of 3%, and when applied to ex vivo mouse hearts the accuracy is 6%, making the LMRS assay comparable to the typical destructive bioanalytical assay.