Accurate determination of protein concentration in the aqueous humor is important in the interpretation of experiments relevant to the pathogenesis and therapy of glaucoma. We made preliminary observations that the high concentration of ascorbic acid in aqueous humor might interfere with commonly used protein determination assays. Therefore, we tested the effect of ascorbic acid upon absorbance in the bicinchoninic acid (BCA), Lowry, and Bradford assays. Ascorbic acid and bovine serum albumin, alone and in combination, were added to BCA, Lowry, and Bradford reagents in 96-well plates. Absorbance was measured at 562 nm (BCA) and 590 nm (Lowry and Bradford). As little as 40 ng and 28 mug of ascorbic acid (typical aqueous humor concentration is 200-400 mug/ml) altered absorbance in the BCA and Lowry assays, respectively. The Bradford assay was not affected by ascorbic acid. Because of the high ascorbic acid and low protein concentration in aqueous humor, assays such as the BCA and Lowry, which are dependent upon oxidation-reduction reactions, must be used with caution. In most situations, the Bradford method is best for determination of aqueous humor protein concentration.