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A method was developed to determine the total amount of biotin present in biotinylated protein conjugates. Conjugates of bovine serum albumin, alkaline phosphatase, and horseradish peroxidase were used in this case study. The extent of biotinylation was determined by complete acid hydrolysis or by enzymatic digestion using proteinase K to release biotin from the biotinylated proteins, followed by sensitive detection of biotin using a coupled HPLC-binding assay system. This detection system is based on the enhancement of the fluorescence of streptavidin-FITC by biotin. The extent of biotinylation determined by this method was compared with the values obtained by a conventional colorimetric method that is based on the displacement of the dye 4-hydroxyazobenzene-2-carboxylic acid (HABA) from the binding sites of avidin. It was found that, because the described method determines the amount of liberated biotin after hydrolysis, it does not suffer from steric hindrance problems associated with the ability of biotin on a protein surface to displace HABA from avidin. Therefore, this method can provide a more accurate determination of the extent of biotinylation. It was also determined that the acid hydrolysis of the biotinylated protein was more effective in releasing the conjugated biotin compared to enzymatic digestion by proteinase K.