Objectives: Comparison of two methods for measuring free hemoglobin in plasma. Study Design: Cross-sectional survey. Material and Methods: A total of 222 samples of human lithium heparin plasma were analyzed, of which 192 samples were from patients undergoing ECMO support, while the remaining 30 were samples with visible hemolysis caused by incorrect sampling of venous blood. Samples from patients undergoing ECMO support were stored at -20 °C for a period not exceeding 6 months. Samples from randomly selected patients who did not undergo ECMO support after finishing routine analysis were prepared for free hemoglobin analysis which was performed on the same day. After the pre-analytical phase, which is the same for both methods, 200 µL of plasma was prepared for each of the methods and the serum indices were first measured on an automated biochemical analyzer, and after that the sample was prepared for measuring free hemoglobin using the Harboe method on a spectrophotometer and after that analysis was performed. The analysis of all 222 samples was done in the month of June 2022. Results: The research results showed that the method of measuring the hemolysis index on an automated biochemical analyzer, although faster, is not reliable enough to replace the currently used Harboe method. Icterus, which is very common in samples from patients undergoing ECMO support, represents an interference that significantly affects the measurement results. Due to the spectral overlap of hemoglobin and bilirubin, the assessment of the degree of icterus in the presence of hemolysis or the degree of hemolysis in the presence of icterus is the most difficult challenge for the measurement of serum indices, as this research has shown. Conclusion: As there is still no recommended method for measuring serum indices or guidelines, we must look for answers to these open questions in future research with the cooperation of laboratories and clinics and adequate support from manufacturers.