OBJECTIVE: This study aimed to assess the diagnostic accuracy of a novel semiautomated method to calculate relative myocardial wall uptake (RCU) of 99mTechnetium-pyrophosphate SPECT/CT in suspected cardiac transthyretin amyloidosis (ATTR). METHODS: Prospective analysis of 108 participants with suspected ATTR. Using novel software, we quantified radiopharmaceutical uptake in the wall of each chamber of the heart at 3 h, calculating RCU for combined ventricular (RCU-V) and whole-heart (RCU-W) measures as a ratio over physiological blood-pool activity. Optimal RCU cut-off ratios were determined using a documented clinical diagnosis of ATTR amyloidosis as the reference standard. RESULTS: The RCU-V method with a cut-off ratio of 1.2 achieved a sensitivity of 100% [95% confidence interval (CI): 90-100%], specificity of 99% (95% CI: 95-100%), and accuracy of 99% (95% CI: 96-100%). The RCU-W method required a higher cut-off ratio of 1.4 to achieve a comparable specificity of 99%. CONCLUSION: This novel SPECT/CT quantification software using the RCU-V method significantly improves diagnostic accuracy for cardiac ATTR and represents an advance compared to existing planar nuclear imaging assessment techniques.