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Abstract Background Total hemoglobin (tHb) measurement is indispensable for determining the patient’s condition (hemorrhagic vs. ischemic) and need for blood transfusion. Conductivity- and absorbance-based measurement methods are used for blood gas analysis of tHb. For conductivity-based measurement, tHb is calculated after converting blood conductivity into a hematocrit value, whereas absorbance measurement is based on light absorbance after red blood cell hemolysis. Due to changes in plasma electrolytes and hemolysis, there is a possibility that conductivity- and absorbance-based measurement methods may cause a difference in tHb. Methods In this study, test samples with controlled electrolyte changes and hemolysis were created by adding sodium chloride, distilled water or hemolytic blood to blood samples collected from healthy volunteers, and tHb values were compared between both methods. Results Conductivity-based measurement revealed reduced tHb value (from 15.49 to 13.05 g/dl) following the addition of 10% sodium chloride, which was also reduced by the addition of hemolysate. Conversely, the addition of distilled water significantly increased tHb value than the expected value. In the absorbance method, there was no significant change in tHb value due to electrolyte change or hemolysis. Conclusions We have to recognize unexpected conductivity changes occur at all times when tHb is measured via conductivity- and absorbance-based measurement methods. The absorbance method should be used when measuring tHb in patients with expected blood conductivity changes. However, when using this method, the added contribution of hemoglobin from hemolytic erythrocytes lacking oxygen carrying capacity must be considered. We recognize that discrepancy can occur between conductivity- and absorbance-based measurement methods when tHb is measured.