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The Duke Criteria have shaped the way infectious endocarditis (IE) is diagnosed in the last 30 years. This study aims to evaluate their current validity and importance in the diagnostic of IE. A retrospective cohort study was conducted on 163 consecutive patients who presented at the University Hospital in Ulm (Germany) with clinical suspicion of IE between 2009 and 2019. With patients' medical records we differentiated between definitive endocarditis (DIE), possible endocarditis (PIE) and rejected endocarditis (RIE) and assessed the validity of the Duke Criteria in comparison to the final discharge diagnosis. We then tried to identify new potential parameters as an addition to the current valid Duke Criteria. The validity of the Duke Criteria improves with the length of hospitalization (especially cardiac imaging criterion, RIE 33.3%, PIE 31.6% and DIE 41.9%, p = 0.622 at admission and RIE 53.3%, PIE 68.4%, DIE 92.2%, p < 0.001 at discharge). At admission, overall sensitivity and specificity were respectively 29.5 and 91.2% in the DIE group. At discharge, sensitivity in the DIE group rose to 77.5% and specificity decreased to 79.4%. Of all screened metrics, microhematuria (p = 0.124), leukocyturia, (p = 0.075), younger age (p = 0.042) and the lack of rheumatoid disease (p = 0.011) showed a difference in incidence (p < 0.2) when comparing DIE and RIE group. In multivariate regression only microhematuria qualified as a potential sixth minor criterion at admission. Even with the latest technological breakthroughs our findings suggest that the Duke Criteria continue to hold value in the accurate assessment of IE. Future efforts must shorten the time until diagnosis., (© 2024. The Author(s).)