Introduction: For interpretation of the most common biochemical markers reference intervals (RIs) are widely considered and reported. Therefore, in this study, we aimed to determine age-specific RIs for some biochemical tests by removing and/or limiting critical gaps including sex, age group, geographical region, measurement device, and type of kits and reagents for routine laboratory biochemical variables in healthy adolescent girls aged 12-19 years.Materials and Methods: In this cross-sectional study a total of 1026 adolescent girl was recruited. According to the study aim, the levels of serum for some routine biochemical tests were measured on a BT-3000 auto-analyzer. On the basis of the non-parametric rank method and by use of CLSI Ep28-A3 guidelines, we determined age-specific RIs, including confidence interval 90%, under specific exclusion criteria.Results: Age partitioning was required for none of the upper and lower limits of biochemical markers. All Serum biochemical tests concentration remained relatively constant throughout the age range. Conclusion: By removing and/or limiting critical gaps involved in reference values of biochemical tests, it has provided an important understanding of metabolic processes and it has facilitated clinical application to track different diseases better