Nephropathy is characterized by urinary micro albumin. Rats are usually used in experimental studies. But, there was no specific and simple method for detecting rat urinary albumin. A specific, easily performed, and sensitive method for quantitatively determining rat urinary albumin is needed. Using rabbit anti-rat albumin polyclonal antibody, biotinylated goat anti-rabbit IgG, avidin conjugated peroxidase, and TMB (3,3',5,5'-tetramethylbenzidine) as substrate, a competitive ELISA for rat albumin in urine was developed. With this method, the urinary albumin in diabetic and normal rats was detected. This method was sensitive to 30 ng/mL and reproducibly quantifies rat urinary albumin levels in the range of 0.05-5 microg/mL. The rabbit anti-rat albumin polyclonal antibody showed no cross reaction with bovine and human serum albumins and rat IgG, and showed little cross-reaction with mouse albumin. The intra-assay CV was less than 10%. The urinary albumin markedly increased in diabetic rats. Since quantifying urinary albumin was very important in the experimental study of diabetic nephropathy, the results from our experiments indicated that this competitive ELISA could be used for quantification of rat urinary albumin.