In this automated procedure for quantifying biotinidase activity in human serum, a manual colorimetric method that measures conversion of the enzyme's artificial substrate N-biotinyl p-aminobenzoate was modified for use with a Technicon AutoAnalyzer II. The intra-run replicate precision (CV) was 2.1% and the day-to-day CV was 4.6% for quality-control sera. Results were linearly related to biotinidase activity in serum over the complete range of clinically relevant values, 0.2 to 11.0 U/L. Moreover, results of the automated assay were not significantly different from those of the manual assay. Because the automated procedure is faster and more precise, we recommend it for population-based studies and some screening studies.