Abstract We have developed a nanogram-level quantitative protein assay based on the binding of colloidal gold to proteins adhered to nitrocellulose paper. The protein–gold complex produces a purple color proportional to the amount of protein present, and the intensity of the stain is quantified by densitometry. Typical assays require minimal starting material (10–20 μl) containing 1 to 5 μg protein. A small volume (2 μl) of protein solution is applied to nitrocellulose paper in a grid array and dried. The nitrocellulose is incubated in colloidal gold suspension with gentle agitation (2–16 h), rinsed with water, and scanned. Densitometric analysis of the scanned images allows quantitation of the unknown sample protein concentration by comparison with protein standards placed on the same nitrocellulose grid. The assay requires significantly less sample than do conventional protein assays. In this report, the Golddots assay is calibrated against weighed protein samples and compared with the Pierce Micro BCA Protein Assay Kit. In addition, the Golddots assay is evaluated with several known proteins with different physical properties.