Objectives Assessment of children’s laboratory test results requires consideration of the extensive changes that occur during physiological development and result in pronounced sex- and age-specific dynamics in many biochemical analytes. Pediatric reference intervals have to account for these dynamics, but ethical and practical challenges limit the availability of appropriate pediatric reference intervals that cover children from birth to adulthood. We have therefore initiated the multi-center data-driven PEDREF project (Next-Generation Pediatric Reference Intervals) to create pediatric reference intervals using data from laboratory information systems. Methods We analyzed laboratory test results from 638,683 patients (217,883–982,548 samples per analyte, a median of 603,745 test results per analyte, and 10,298,067 test results in total) performed during patient care in 13 German centers. Test results from children with repeat measurements were discarded, and we estimated the distribution of physiological test results using a validated statistical approach ( kosmic ). Results We report continuous pediatric reference intervals and percentile charts for alanine transaminase, aspartate transaminase, lactate dehydrogenase, alkaline phosphatase, γ-glutamyl-transferase, total protein, albumin, creatinine, urea, sodium, potassium, calcium, chloride, anorganic phosphate, and magnesium. Reference intervals are provided as tables and fractional polynomial functions (i.e., mathematical equations) that can be integrated into laboratory information systems. Additionally, Z -scores and percentiles enable the normalization of test results by age and sex to facilitate their interpretation across age groups. Conclusions The provided reference intervals and percentile charts enable precise assessment of laboratory test results in children from birth to adulthood. Our findings highlight the pronounced dynamics in many biochemical analytes in neonates, which require particular consideration in reference intervals to support clinical decision making most effectively.