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Background/Objectives: Limb lengthening and deformity correction techniques, particularly distraction osteogenesis, have significantly evolved in pediatric orthopedics. This study examines the temporal changes of key biochemical markers—vascular endothelial growth factor (VEGF), fibroblast growth factor 1 (FGF-1), and the propeptide of type I collagen (P1NP)—during the limb lengthening process. Methods: Twenty pediatric patients (aged 13–16) underwent distraction osteogenesis using the Circular Hexapod External Fixator. Peripheral blood samples were collected pre-treatment, three weeks after initiating distraction, and one month post-lengthening. Marker levels were measured using ELISA. Results: Serum VEGF concentrations significantly increased during treatment, peaking at T2 (T1 35.91 ± SD 5.54 vs. T2 293.47 ± SD 69.57, p < 0.0001), then declined at T3 (293.47 ± SD 69.57 vs. 40.86 ± SD 6.26, p < 0.0001). FGF-1 showed minor fluctuations initially but significantly increased by T3 (18.14 ± SD 4.57 vs. 41.56 ± SD 17.15, p < 0.01), about 2.3 times higher than baseline. P1NP concentrations exhibited a linear increase, with a significant rise from T2 to T3 (234.06 ± SD 36.57 vs. 280.68 ± SD 35.63, p < 0.05), while the T1 to T2 increase was not statistically significant, indicating ongoing osteoblastic activity and bone formation. Conclusions: This study highlights the dynamic changes in VEGF, FGF-1, and P1NP during distraction osteogenesis, emphasizing their roles as biomarkers of bone regeneration. These findings enhance the understanding of bone healing mechanisms and could inform future therapeutic strategies for pediatric limb lengthening. Further research is warranted to explore their clinical utility. [ABSTRACT FROM AUTHOR]