BACKGROUND: An enzymatic kit method for the determination of serum creatinine was optimized for use with OLYMPUS AU 400 Auto analyzer and its performance characteristics and practicability compared with kinetic Jaffe-based method. Effects of some common interfering substances like glucose and bilirubin on the kinetic Jaffe's and the enzymatic methods were compared. Method comparison between the enzymatic creatinine method (y) and Jaffe's kinetic method (x) gave the following equation for the normal group: y=0.97*x+0.0 and a coefficient correlation R of 0.98.There was very good agreement between both the methods as intra class correlation coefficient (ICC) was between 0.81-1.In the presence of high levels of glucose or bilirubin, the coefficient correlation values were 0.99 and 0.89respectively.