In this study, we aim to apply a split-luciferase bioluminescence assay to functionally assess <i>COL4A3</i>, <i>COL4A4</i>, or <i>COL4A5</i> variants of uncertain significance (VUS) identified in pediatric patients with Alport syndrome, and to explore its value in supporting variant interpretation and diagnostic evaluation.<h4>Methods</h4>A retrospective analysis included 31 children who met established clinical and pathological diagnostic criteria for Alport syndrome, but in whom genetic testing identified VUS in <i>COL4A3</i>, <i>COL4A4</i>, or <i>COL4A5</i>. This evidence concerns the gene COL4A5 and Alport syndrome.