The coomassie brilliant blue (CBB) protein assay first developed by Bradford became an alternative for an ever-increasing number of researchers because of its simplicity, rapidity and being free from interference by some common laboratory reagents which have been shown to affect the lowry assay. Despite all its advantages, many problems have been encountered by researchers in using this procedure. One of the main drawbacks of this procedure is the nonlinearity which has been noted since Bradford developed the CBB-protein assay. The effect of ingredients in color-developing reagent on the absorbances at 465 nm and 595 nm, of free CBB and protein-binding CBB mixture was tested respectively. The results showed that the main factor which affects the linearity of calibration curve is H⁺ concentration. A new color-developing reagent formula which consists of CBB, HCl and NaCl, may improve the nonlinearity.