The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains an unusual amino acid, hypusine [N (ε)-(4-amino-2-hydroxybutyl)-lysine]. eIF5A and its hypusine/deoxyhypusine modification are vital for eukaryotic cell proliferation. Hypusine is formed posttranslationally by two enzymatic steps catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. Deoxyhypusine hydroxylase catalyzes a stereo-specific hydroxylation of the deoxyhypusine residue in the eIF5A intermediate protein, eIF5A(Dhp). The enzyme is totally specific for this protein and does not act on short peptides (<50 amino acids). The assay measures the conversion of the radiolabeled deoxyhypusine residue to a hypusine residue in eIF5A. Optimum conditions for the reaction and two detection methods for the product, hypusine-containing eIF5A, are described in this chapter. The first, and most reliable, method is the measurement of the amount of [(3)H]hypusine in the protein hydrolysate after its separation from [(3)H]deoxyhypusine, by ion exchange chromatography. This method does require specialized equipment. The second method is based on counting the total TCA soluble radioactivity after sodium periodate oxidation of the reaction mixture, since the radiolabeled 4-amino-2-hydroxy butyl moiety of the hypusine residue is cleaved and is released from protein as radiolabeled β-propionaldehyde and formaldehyde by periodate oxidation.