BACKGROUND: Smith-Lemli-Opitz Syndrome (SLOS) is an autosomal recessive disorder characterized by specific clinical features. This study investigated the correlation and transference ratio of 7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) as screening/diagnostic markers for SLOS in whole blood and plasma samples. METHODS: The gas chromatography-mass spectrometry (GC-MS) method was employed to analyze whole blood and plasma samples collected from 28 healthy volunteers and one patient with SLOS. Before analysis, an optimized and validated assay for 7-DHC and CHOL in whole blood was developed. Pearson's correlation analysis was then performed to assess the correlation between the levels of 7-DHC and CHOL in whole blood and plasma. RESULTS: The correlation analysis revealed a significant correlation between the levels of 7-DHC and CHOL in whole blood and plasma, with correlation coefficients (r) greater than 0.5 (0.565 for 7-DHC and 0.692 for CHOL). These findings suggest the feasibility of transference between whole blood and plasma samples. CONCLUSIONS: Whole blood can be considered an optimal alternative diagnostic tool for predicting SLOS in carriers, surpassing the reliance on plasma alone. This study introduces a new diagnostic matrix for determining 7-DHC and CHOL, expanding the scope of SLOS diagnosis and research.