Ulva lactuca L. is a green alga of the Ulvaceae family distributed worldwide, which has gained increased attention due to its high reproductive rate, which leads to the phenomenon known as "green tides", accelerated by water eutrophication. While algae from the Ulvaceae family are used for food and feed purposes, their biomass currently has limited applications. However, the cell wall of these algae is rich in polysaccharides, of which ulvan is the most peculiar and interesting. Ulvan is composed of disaccharides repeating units, constituting in rhamnose-3-sulfate (Rha3S) linked to D-glucuronic acid (GlcA), L-iduronic acid (IduA) or Dxylose (Xyl) by predominantly beta glycosidic bonds. Ulvan oligosaccharides has been associated with anticholesterolemic and antioxidant activities, making the production of these compounds a promising opportunity for biomass utilization1. Since these bonds are highly resistant to conventional acidic hydrolysis methods, enzymatic hydrolysis is preferred to ensure efficiency and the production of food-grade products. To this end, ulvan lyases (EC 4.2.2), found in marine bacteria, can be used for the catalysis of the endolithic cleavage between Rha3S and uronic acids, by a β-elimination reaction2. Aim of the present study was the optimization of the enzymatic hydrolysis of ulvan to obtain a pool of ulvan oligosaccharides to be testet for biological activities. Ulvan were extracted from U. lactuca collected in an aquaculture tank of Orbetello lagoon. A recombinant ulvan lyase of PL24 family commercially available was used for the hydrolysis. The enzyme was characterized kinetically and different concentrations of buffer medium (composed of NaCl and Trizma® base both from 0 to 100 nM) were tested to minimize the residual presence of salts in the final products. The activity of the enzyme was measured for 24 h as spectrophotometric absorbance at 235 nm (indication of double bonds formation after β-elimination), 1H-NMR spectra were acquired to monitor the production of oligosaccharides, and Dynamic Light Scattering measurements were applied to evaluate the hydrodynamic volume of the hydrolyzed ulvan in solution. In summary, ulvan lyase resulted a robust enzyme able to rapidly and extensively hydrolyze ulvan at different composition of the medium. To provide insights into the impact of ulvan on gut health and to promote future uses of Ulva biomass, the hydrolyzed ulvan will be tested in vitro for its prebiotic activity.