Abstract Background Single-sample (screening) rule-out of acute myocardial infarction (AMI) with troponin requires derivation of a single-test screening threshold. In data sets with small event numbers, the lowest one or two concentrations of myocardial infarction (MI) patients dictate the threshold. This is not optimal. We aimed to demonstrate a process incorporating both real and synthetic data for deriving such thresholds using a novel pre-production high-precision point-of-care assay. Methods cTnI concentrations were measured from thawed plasma using the Troponin I Next (TnI-Nx) assay (i-STAT; Abbott) in adults on arrival to the emergency department with symptoms suggestive of AMI. The primary outcome was an AMI or cardiac death within 30 days. We used internal–external validation with synthetic data production based on clinical and demographic data, plus the measured TnI-Nx concentration, to derive and validate decision thresholds for TnI-Nx. The target low-risk threshold was a sensitivity of 99% and a high-risk threshold specificity of >95%. Results In total, 1356 patients were included, of whom 191 (14.1%) had the primary outcome. A total of 500 synthetic data sets were constructed. The mean low-risk threshold was determined to be 5 ng/L. This categorized 38% (95% CI, 6%–68%) to low-risk with a sensitivity of 99.0% (95% CI, 98.6%–99.5%) and a negative predictive value of 99.4% (95% CI, 97.6%–99.8%). A similarly derived high-risk threshold of 25 ng/L had a specificity of 95.0% (95% CI, 94.8%–95.1%) and a positive predictive value of 74.8% (95% CI, 71.5%–78.0%). Conclusions With the TnI-Nx assay, we successfully demonstrated an approach using synthetic data generation to derive low-risk thresholds for safe and effective screening.