OBJECTIVE The aim of this study was two-fold: Firstly, to compare the serum creatinine concentration measured by enzymatic method and uncompensated kinetic Jaffe method, and secondly, to compare the effects of certain interfering substances such as glucose, bilirubin, proteins, triglycerides and hemoglobin on creatinine measurement. METHODS The determination of serum creatinine concentrations by enzymatic method and uncompensated kinetic Jaffe method was performed on ADVIA® 2400 analyzer. The interfering substances were tested by adding solutions of interference to serum pool with low (62 μmol/L), medium (221 μmol/L) and elevated (486 μmol/L) creatinine concentration. RESULTS In the method comparison study, despite the fact that the correlation between both methods for determining serum creatinine is very good (r = 0.998, p < 0.001), the regression analysis revealed that the results are not transferable, as indicated by the slope and intercept, which are significantly different from 1 and 0, respectively. A positive bias of + 14.1% in the determination of serum creatinine by uncompensated kinetic Jaffe method was found, and when the creatinine value is lower than ∼ 180 μmol/L this difference or bis progressively increases. We found a significant positive interference due to proteins and glucose and a significant negative interference due to bilirubin by kinetic Jaffe method, and no interferences by enzymatic method were found. CONCLUSIONS In conclusion, the enzymatic method is the best choice for determining serum creatinine with the ADVIA® 2400 analyzer.