COL4A4 and Alport syndrome: When the luminescence intensity was below 397.8, the sensitivity for distinguishing VUS from WT was 96.77%, and the specificity was 100%.<h4>Conclusions</h4>The split-luciferase bioluminescence assay was successfully applied as an <i>in vitro</i> functional approach to assess <i>COL4A3</i>, <i>COL4A4</i>, or <i>COL4A5</i> VUS, and may reliably assist in the diagnostic evaluation of pediatric patients with suspected Alport syndrome whose genetic testing reveals VUS, particularly in cases where pathological assessment is unavailable or not feasible.