Objectives To evaluate the analytical characteristics of a novel high-sensitivity cardiac troponin T (hs-cTnT) test on the automatic light-initiated chemiluminescent assay (LiCA®) system, and validated its diagnostic performance for non-ST-segment elevation myocardial infarction (NSTEMI). Methods Studies included an extensive analytical evaluation and established the 99th percentile upper reference limit (URL) from apparently healthy individuals, followed by a diagnostic performance validation for NSTEMI. Results Sex-specific 99th percentile URLs were 16.0 ng/L (1.7 % CV: coefficient of variation) for men (21–92 years) and 13.4 ng/L (2.0 % CV) for women (23–87 years) in serum, and 30.6 ng/L (0.9 % CV) for men (18–87 years) and 20.2 ng/L (1.4 % CV) for women (18–88 years) in heparin plasma. Detection rates in healthy individuals ranged from 98.9 to 100 %. An excellent agreement was identified between LiCA® and Elecsys® assays with a correlation coefficient of 0.993 and mean bias of −0.7 % (−1.8–0.4 %) across the full measuring range, while the correlation coefficient and overall bias were 0.967 and −1.1 % (−2.5–0.3 %) for the lower levels of cTnT (10–100 ng/L), respectively. At the specific medical decision levels (14.0 and 52.0 ng/L), assay difference was estimated to be Conclusions LiCA® hs-cTnT is a reliable 3rd-generation (level 4) high-sensitivity assay for detecting cardiac troponin T. The assay is acceptable for practical use in the diagnosis of NSTEMI.