CAD is a large, 2,225 amino acid multi-enzymatic protein required for de novo pyrimidine biosynthesis. Pathological CAD variants cause a developmental and epileptic encephalopathy which is highly responsive to uridine supplements. CAD deficiency is difficult to diagnose because symptoms are non-specific, there is no biomarker, and the protein has over 1,000 known variants. To improve diagnosis, we assessed the pathogenicity of 20 unreported missense CAD variants using a growth complementation assay that identified 11 pathogenic variants in 7 affected individuals; they would benefit from uridine treatment. We also tested 9 variants previously reported as pathogenic and confirmed the damaging effect of 7.