Abstract We describe a simple assay for small amounts of protein that is insensitive to sodium dodecyl sulfate (SDS) or many common interfering substances including Tris and reducing sugars. For this reason, it is particularly useful in the analysis of protein content of samples prior to SDS electrophoresis. The assay consists of the following steps: (i) absorption of protein to nitrocellulose; (ii) fixation of the bound protein with methanol; (iii) staining of the bound protein with amido black; and (iv) elution and spectrophotometric measurement of the bound dye. The assay is sensitive to as little as 0.5 μg of protein in 1 μl of solution. Although SDS does not interfere appreciably with measurement, Nonidet-P40 does.