Ictal single photon emission computed tomography (SPECT) is a powerful tool for noninvasive seizure localization, but it has been underutilized because of practical challenges, including difficulty in implementing ictal-interictal SPECT difference analysis. We previously validated a freely available utility for this purpose, ictal-interictal subtraction analysis by statistical parametric mapping (SPM) (ISAS). To further simplify and improve the difference imaging technique, we now compare a new algorithm, ISAS BioImage Suite (see http://spect.yale.edu and http://bioimagesuite.org), to the original ISAS method in 13 patients with known seizure localization. We found that ISAS BioImage Suite was in agreement with the original algorithm in all cases for which ISAS correctly identified a single unambiguous region of seizure onset. We also tested for possible effects of scan-order bias in the control group used for the analysis and found no significant effect on the results. These findings establish a simple, validated and objective method for analyzing ictal-interictal SPECT difference images for use in the care of patients with epilepsy.