The molecular diagnostic workflow, herein described, represents a first-line tool to identify simultaneously the two Italian PVs hot spot in patients with suspicion of IIH but doesn’t give a high risk of false negative results; in fact, if the samples result as negative for the targeted variants in the this first screening step, our workflow provides for sequencing of all coding and flanking intronic regions of CYP24A1. Here, CYP24A1 is linked to pseudotumor cerebri.