Background: Biotinidase deficiency is an autosomal recessive disorder of biotin metabolism that can be effectively managed when detected early through newborn screening. Biotinidase activity is influenced by neonatal and preanalytical factors, which may affect the interpretation of screening results. Following the introduction of biotinidase deficiency screening in Mongolia in 2025–2026, population-specific data are needed to support accurate screening interpretation. Aim: To determine the distribution and percentile values of biotinidase activity in Mongolian newborns and evaluate associated neonatal and preanalytical factors. Materials and Methods: This analytical cross-sectional study included 6,764 newborns from 12 healthcare facilities in Mongolia between August 2025 and May 2026. After excluding initial screen-positive samples and those with transport times exceeding 30 days, 6,258 newborns were included in the primary analysis. Biotinidase activity was measured in dried blood spots using the Neonatal Biotinidase 3018-0010 kit and VICTOR2D analyzer. Percentile curves by gestational age and birth weight were smoothed using LOWESS. Group differences and associations were assessed using Welch ANOVA and Pearson correlation, respectively. Result: Among 6,258 newborns, mean biotinidase activity was 133.3±32.6 U, with a median of 130.2 U and a P2.5-P97.5 interval of 78.4-206.6 U. Median activity tended to increase with gestational age and birth weight, while the smoothed percentile curves remained above the 58.5 U screening cutoff. Mean activity increased from 124.4±34.1 U in samples collected before 24 hours to 149.9±44.5 U in those collected after 96 hours (p<0.001). Activity showed weak positive correlations with gestational age (r=0.042, p=0.001), birth weight (r=0.114, p<0.001), and body length (r=0.102, p<0.001), but negative correlations with time from sample collection to laboratory receipt (r=−0.327, p<0.001) and from laboratory receipt to testing (r=−0.154, p<0.001). Conclusion Population-specific percentile values for biotinidase activity were established in a large sample of Mongolian newborns. The findings provide baseline data for interpreting biotinidase activity and highlight the importance of standardized sample collection, transport, and testing.