A self-prepared benzethonium chloride reagent for determination of protein in urine and cerebrospinal fluid (CSF) was prepared and evaluated. The accuracy, precision, detection limit, linearity and stability of the reagent were studied on a Hitachi 704 automatic analyzer. For accuracy, the average recovery of the self-prepared reagent and the commercial reagent were 95.4% and 98.0% respectively. The precision was evaluated by analyzing two levels of urine control. It was found that the within-run and the between-run coefficients of variation (%CV) of the self-prepared reagent were 1.8% to 3.9% and 1.8% to 3.8% respectively, whereas those of the commercial reagent were 1.4% to 3.9% and 1.5% to 3.8% respectively. The selfprepared reagent and the commercial reagent gave the same detection limit and linearity of 2.0 mg/dL and 200 mg/dL respectively. The correlation (r) of protein determination in 200 urine and CSF patient samples was 0.9990 (y = 0.996X + 0.356) with no significant difference (p > 0.05) found. A self-prepared reagent was stable for at least 6 months when stored in a refrigerator at 4 oC. The cost per test of this reagent is about 300-fold less than that of the commercial reagent. In conclusion, the self-prepared reagent gave satisfactory score for protein determination, and was determined to be as good as a commercial reagent and is applicable for routine protein determination in the clinical chemistry laboratory.