Smith-Lemli-Opitz syndrome (SLOS; MIM 270400) is a genetic disorder characterized by hypocholesterolemia and elevated 7-dehydrocholesterol (7DHC) levels resulting from mutations affecting 7-dehydrocholesterol reductase. We describe a colorimetric assay for 7DHC with potential application to large-scale screening for SLOS. Reaction of 7DHC and its esters with the Liebermann-Burchard reagent resulted in a brief initial absorbance at 510 nm (pink color) followed by an absorbance at 620 nm (blue color) after 2 min, while cholesterol samples were essentially colorless. The assay could identify typical SLOS blood samples by their pink color and increased absorbance at 620 nm after 2 min. Colorimetric identification of mild SLOS cases requires monitoring of the transient absorbance at 510 nm, which must be detected immediately after rapid, consistent mixing of the reagents. The need for special mixing devices and rigorous validation precludes sporadic use of the assay for diagnosing suspected SLOS cases. We also studied the stability of 7DHC in dried SLOS blood spots on Guthrie cards, which are widely used for archiving neonatal blood. Decomposition of 7DHC was effectively retarded by storage at low temperature and by precoating of the cards with antioxidants. The combined results provide a foundation for development of a simple, automated test for SLOS screening.