Objective:The objective of the study was to determine the cutoff points (CP) of nine neonatal screening biomarkers performed at the Laboratory of the Center for Neonatal and Genetic Studies in Mexico City, comparing them with the CP recommended by the manufacturer of the reagents. Materials and methods: A descriptive cross-sectional research was carried out. The study included 2805 newborns aged 1-30 days, who had a drop of dried whole blood collected on filter paper, then quantified for their concentrations of thyroid-stimulating hormone, thyroxine, phenylalanine, immunotrypsin, galactose-1-phosphate uridyltransferase, galactose, glucose-6-phosphate dehydrogenase, biotinidase, and 17-alpha hydroxyprogesterone, by immunofluorescence method (DELFIA, PerkinElmer LifeSciences time-resolved fluorometry). The cutoff point (excluding atypical values) was calculated with the data obtained for each test, and the mean and standard deviation were determined. Sensitivity and specificity were determined with receiver operating characteristic (ROC) curves. The determined CPs were compared with the CPs recommended by the manufacturer of the reagents. Results: Except for T4, it was shown that, when comparing the CP obtained, these were lower compared to the CPs recommended by the manufacturer. Conclusion: The CPs obtained in Mexican neonates differ from those recommended by the manufacturer. In addition, CPs are analytically acceptable, as they have high sensitivity and specificity. Consequently, these CPs can be used as a reference in clinical practice for the possible early detection of metabolic diseases in newborns.