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Introduction The availability of pediatric age- and sex-matched reference curves for bone markers is essential for appropriate assessment of bone turnover and treatment follow-up of bone disorders. The aim of this work was to obtain updated reference equations for collagen type I C-telopeptides (CTX-1) and bone alkaline phosphatase (BAP) by using an alternative statistical approach. The study included 1502 Italian pediatric subjects from 6 months to 16 years of age (686 females and 816 males) subjected to CTX-1 and BAP measurement during a six-year period. Methods The unselected population of patients was used for the calculation of age- and sex-matched reference curves by using a multivariate statistical analysis after an appropriate validation with a selected population of 184 healthy subjects (6 months-16 years; 88 females and 96 males). The effect of age, sex, puberty based on Tanner stage evaluation and anthropometrics on the variations of the two bone markers was then studied. Results Pediatric reference curves were obtained for CTX-1 and BAP from 3465 results retrieved by our Laboratory Information System. The equations for the calculation of reference values were obtained for boys and girls. The two bone markers markedly varied according to age, sex and pubertal stage with females displaying higher values during Tanner stages II and III and males during stages III and IV. Conclusions The application of a novel statistical approach provided reference curves for CTX-1 and BAP. This method, moreover, could be applied in pediatrics to obtain reference intervals for other biomarkers.