Background <p>Accurate assessment of biological maturity (BM) is essential in pediatric sports medicine to support individualized training and injury prevention. Traditional methods, such as sexual maturity ratings (SMR) and radiographic imaging, have limitations including subjectivity, discomfort, and radiation exposure. This study aimed to evaluate and validate the use of two established ultrasonography protocols for assessing biological maturity at the tibial tuberosity in healthy, physically active youth.</p> Methods <p>Seventy-three children and adolescents (41 boys, 32 girls; mean age 13.4 years) were recruited from schools in Northern Sweden. BM was assessed using SMR and two established ultrasonographic protocols (Sailly and Kijima). Validity was evaluated using Spearman’s rho, and inter- and intrarater reliability were assessed with Cohen’s weighted kappa.</p> Results <p>Ultrasonographic assessments showed strong agreement with SMR-based evaluations. Interrater reliability was substantial for both methods (κ = 0.761–0.814, <i>p</i> &lt; 0.001). The method proved feasible for use by trained physiotherapists and did not require radiological or endocrinological expertise.</p> Conclusion <p>Ultrasonography, combined with anthropometric data, is a valid, reliable, and practical alternative for assessing BM in youth. Its non-invasive, radiation-free nature and scalability make it particularly suitable for pediatric and sports settings. Further research should explore its application in diverse populations and its role in guiding biologically informed training strategies.</p>