<p>This study was conducted to evaluate the accuracy and reliability of the Spectro-DPRA, an enhanced method for the OECD TG 442C (direct peptide reactivity assay, DPRA), serving as an alternative to animal testing for skin sensitization. The validation of Spectro-DPRA was executed across four participating laboratories (Lab 1, Lab 2, Lab 3, and Lab 4, the latter only participating in the proficiency test) adhering to GLP principles. It covered transferability, proficiency, within laboratory and between laboratory reproducibility tests, and a predictive capacity test using 40 additional substances. In tests with 10 proficiency substances, results from all participating laboratories fell within the respective reference ranges for 8 of the 10 substances for each peptide. The within laboratory reproducibility for 12 test substances demonstrated success rates of 83.3% for both Lab 1 and Lab 3, and 100% for Lab 2. The between laboratory reproducibility for 20 test substances showed an 85% concordance rate. The predictive capacity for these substances did not align with the test method similarity criteria stipulated in the OECD Series on Testing and Assessment No. 303 (performance standards for the assessment of proposed similar or modified in vitro skin sensitisation DPRA and ADRA test methods, OECD PS No.303), with accuracy, sensitivity, and specificity rates at 74.6%, 70.9%, and 81.4%, respectively. However, the predictive capacity for an additional 40 substances revealed high accuracy (85.2%), sensitivity (82.2%), and specificity (97.0%). Although the Spectro-DPRA did not completely conform to the OECD PS No.303 criteria for test method similarity, it showed high predictive accuracy of over 80% for the 40 additional substances, confirming its reliability as a test method for skin sensitization evaluation. </p>