Accurate quantitation of the protein content of human milk (HM) is clinically important for both determining protein and energy intakes of HM-fed infants. Protein can be determined by Kjeldahl analysis or colorimetric assays. Colorimetric assays are rapid and convenient, but usually overestimate the protein content when compared to Kjeldahl protein (KP). In this study a protein standard based on human milk protein (HMPS) was isolated by size exclusion column chromatography. Purity and composition of the standard was determined by gel electrophoresis, Kjeldahl and amino acid analyses. The protein content of 20 mature milk samples was determined by the Lowry, BCA and BioRad colorimetric assays, using bovine serum albumin, human serum albumin/IgG, and HMPS as standards. These results were compared with KP results using Student's t-test. All colorimetric assays overestimated milk protein content; however, the Lowry assay gave the lowest protein levels, with our protein standard yielding values closest to the KP value.