BACKGROUND: Preanalytical interferences caused by hemolysis, icterus, and lipemia (HIL) remain major sources of diagnostic error in clinical laboratories. Manufacturer-declared cutoffs are often based on limited validation data and may not reflect local analytical performance, particularly across heterogeneous assay systems. This study aimed to determine experimentally derived HIL interference thresholds for a broad panel of clinical chemistry and immunoassay tests and to compare them with manufacturer specifications. METHODS: Nineteen routinely used assays (14 clinical chemistry and 5 immunoassays) were evaluated on the Abbott Architect c16000 and Siemens Advia Centaur XP platforms following CLSI EP07-A2 guidelines. Serum pools were spiked with graded levels of hemolysate, conjugated bilirubin, or lipid emulsion. Bias was calculated at each interference level, and thresholds were defined as the point exceeding the lower of the desirable bias or total allow-able error (TEa) limits or the manufacturer's declared cutoff. RESULTS: Hemolysis produced significant deviations in several assays. Potassium interference began at 0.85 g/L hemoglobin versus the declared 1.25 g/L limit, and HDL-cholesterol exceeded the 5% desirable bias at 2.5 g/L, well below the 7.2 g/L manufacturer threshold. Bilirubin interference affected creatinine and glucose from 124 µmol/L, despite declared limits above 340 µmol/L, consistent with spectral absorption in the Jaffé reaction. Lipemic interference was observed at 2.2 mmol/L for sodium and HDL-cholesterol, nearly fivefold lower than the stated cutoffs. Among immunoassays, ferritin showed a positive bias, TSH a negative shift, and vitamin B12 a bidirectional pattern under hemolytic conditions; fT4 displayed concentration-dependent bias under lipemia. CONCLUSIONS: Manufacturer-declared HIL limits may be overly permissive, masking clinically relevant bias. Locally verified thresholds revealed earlier interference onset for several analytes. The immunoassay platform used lacked an automated HIL index analyzer, highlighting the need to integrate such detection systems for optical and matrix-related interference monitoring. Routine local validation and continuous index surveillance are essential to ensure analytical accuracy and patient safety.