This study compared the results of hemolyzed samples and re-collected samples to investigate a hemolysis influence and an acceptable hemolysis index (HI). Before and after hemolysis, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), amylase (Amy), direct bilirubin (D-bil), total bilirubin (T-bil), creatine phosphokinase (CK), gamma glutamyl transferase (GGT), iron, potassium (K), lactate dehydrogenase (LDH), magnesium (Mg), phosphorus (Phos), total protein (TP), and uric acid (UA) showed significant results in the paired t-test. LDH, K, iron, AST, CK, GGT, TP, Amy and Phos had a high correlation between the degree of hemolysis and the results of samples. When comparing Roche's cut-off HI with HIQChigh obtained using quality control (QC) high standard deviation (SD), AST, D-bil, CK, and LDH were similar, but Amy, GGT, K, iron, Phos, and TP were lower than the cut-off HI of Roche, while ALP and ALT were higher. Some analytes which showed no significant results in the paired t-test, were found to have significant results in HI>200. Hence, it is suggested that the hemolyzed sample should be rejected if HI>200. Based on this study that some analytes were affected when HI<100, we recommend to set the standard of hemolysis starting from HI>50.