High-sensitivity cardiac troponin T (hs-cTnT) assays are crucial in evaluating chest pain, but their increased sensitivity makes them susceptible to pre-analytical interferences. False-positive results may lead to unnecessary clinical interventions, especially in pediatric populations—a 14-year-old male patient presented with chest pain. Two simultaneously collected blood samples showed significantly different hscTnT levels: <5.4 ng/L in the first sample (processed from an aliquot) and 31.7 ng/L in the second (analyzed from the primary tube). Reanalysis of the second sample after centrifugation yielded 45.2 ng/L. However, hs-cTnT measured in the EDTA plasma sample and in a freshly collected sample were both <5.4 ng/L. ECG and echocardiographic findings were normal. The inconsistent hs-cTnT values were attributed to pre-analytical factors such as fibrin clots or gel droplets in the primary tube. These artifacts can interfere with immunoassays and produce falsely elevated results, despite being visually undetectable. The consistent normal results in EDTA plasma and the freshly collected sample confirmed the presence of a preanalytical error. Pre-analytical variability can significantly impact hs-cTnT testing, leading to misleading clinical interpretations. Laboratory professionals and clinicians must collaborate when test results do not align with clinical findings to prevent misdiagnosis and unnecessary intervention.