von Hippel-Lindau (VHL) is an autosomal-dominant tumour susceptibility disorder associated with pathogenic germline variants in the VHL gene that put patients at increased risk of developing benign and malignant tumours within various organs. While current CLIA (Clinical Laboratory Improvement Amendments) genetic tests demonstrate a high detection rate of pathogenic VHL variants, clinical manifestations of VHL with atypical germline alteration undetectable by established genetic tests have been reported by Vocke et al, indicating the need for bespoke methods confirming germline alterations. We CLIA-validated a test using PCR and fragment analysis that evaluates a pathogenic inversion of 291 kb found within intron 2 of VHL and the 3′ UTR of TTLL3 in patients with canonical VHL manifestations and no conventional germline alterations. Validation results matched as expected by achieving 100% concordance. Although clinical diagnosis of VHL is possible in the absence of conclusive pathogenic cause, this report demonstrates bespoke CLIA methodologies are essential for managing hereditary diseases and promoting early symptom detection. Our CLIA assay allows for greater confidence in diagnosis for families dealing with complex structural germline alterations and may aid in future efforts at corrective therapies to ease patient suffering. This work was performed in our CLIA-certified laboratory (CLIA ID # 21D0947274).