Deconvolution of septal penetration (DSP) has been developed to improve quantification so as to allow the use of low-energy high-resolution collimators for iodine 123 cardiac single photon emission computed tomography (SPECT) imaging. The purpose of this study is to optimize its acquisition and processing protocols. Planar images of a 9-compartment phantom loaded with variable radioactive concentrations were acquired to derive optimal scatter compensation scaling factors for 20% and 15% photopeak energy window configurations, respectively. A cardiac phantom, loaded with high and low heart-to-calibration ratios (HCRs), respectively, was imaged with both configurations. Repeated acquisitions were done for medium-energy all-purpose collimators for comparison. Critical frequencies for Butterworth filtering were optimized by use of defect contrast and normal short-axis uniformity as selection indices. HCRs were calculated with planar projection and different reconstruction methods, respectively, and then compared with the true HCRs. SPECT produced more accurate HCRs than planar imaging. With the optimized parameters for scatter compensation and filtering, the 2 energy window configurations yielded similar results. Iterative reconstructions with DSP yielded more accurate HCRs than other reconstructions without DSP. The optimized protocols based on DSP show promise that quantification of I-123 cardiac SPECT imaging can be achieved with the widely available low-energy high-resolution collimators.