Several lines of clinical evidence as well as guidelines and recommendations suggest that the overall diagnostic performance of D-dimer testing outstrips that of any other biomarker in the diagnostic approach of patients with venous thromboembolism or disseminated intravascular coagulation. Along with specific technical characteristics, the analytical performance of each D-dimer immunoassay should, however, be assessed before implementation in clinical practice. The aim of this study was to evaluate the analytical performance of HemosIL AcuStar D-Dimer immunoassay, a novel chemiluminescent immunoassay specifically designed for the instrument Instrumentation Laboratory ACL AcuStar. The within and between-run imprecision (n = 20) was comprised between 3.6 and 5.8%. The linearity was excellent up to 16 200 ng/ml (r = 1.00; P < 0.001), and optimal between 224 and 69 900 ng/ml (r = 0.992; P < 0.001). A significant agreement of values was observed between HemosIL AcuStar D-Dimer and HemosIL D-Dimer HS for ACL TOP (r = 0.884; P < 0.001), as well as with Vidas D-Dimer (r = 0.791; P < 0.001). Results of HemosIL AcuStar D-Dimer displayed a modest negative bias as compared with those of Vidas D-dimer (mean bias -22%; 95% Confidence Interval, -122% to 49%). The analytical accuracy assessed against Vidas D-dimer also yielded an area under the curve of 0.998 (P < 0.001). As regards, the preliminary definition of cutoff value, optimal sensitivity (100%) and specificity (96%) were found at a diagnostic threshold of 466 ng/ml. The results of this investigation attest that the novel HemosIL AcuStar D-Dimer is characterized by remarkable accuracy and precision, optimal linearity and excellent agreement with the reference immunoassay. As such, its technical and analytical performance would make it a suitable method for the rapid and accurate quantification of D-dimer in clinical laboratories.