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Recent studies have reported that biotin interferes with certain immunoassays. In this study, we evaluated the analytical interference of biotin on immunoassays that use streptavidin-biotin in our pediatric hospital. We tested the effect of different concentrations of biotin (1.5–200 ng/ml) on TSH, Prolactin, Ferritin, CK-MB, β-hCG, Troponin I, LH, FSH, Cortisol, Anti-HAV antibody (IgG and IgM), assays on Ortho Clinical Diagnostic Vitros 5600 Analyzer. Biotin (up to 200 ng/mL) did not significantly affect Troponin I and HAV assays. Biotin (up to 12.5 ng/ml) resulted in <10% bias in CK-MB, β-hCG, AFP, Cortisol, Ferritin assays and biotin >6.25 ng/mL significantly affected TSH (>20% bias) assay. Prolactin was significantly affected even at low levels (Biotin 1.5 ng/mL). Thus, we recommend educating physicians about biotin interference in common immunoassays and adding an electronic disclaimer.