Purpose: To evaluate the feasibility of exploiting ultrasonication coupled with ionotropic gelation in order to prepare tripolyphosphate (TPP)-chitosan glutamate nanoparticles suitable for the delivery of the enzyme prolidase. Methods: All the parameters for the preparation of TPP-chitosan nanoparticles in terms of components weight ratio, ultrasonication conditions and time-saving nanoparticles recovery conditions were optimized. The best formulation was loaded with the prolidase. All the nanoparticles were characterized in terms of morphology, size, polydispersity, zeta-potential, yield of the process and encapsulation efficiency. The in-vitro activity of the prolidase was assessed by capillary electrophoresis ( CE). Results and conclusions: A TPP to chitosan weight ratio of 0.2: 1 combined with one ultrasonication cycle (4 min using the probe-type sonifier at 75% power) obtained well-formed nanoparticles of spherical shape, mean size of similar to 365nm (polydispersity index 0.3) and a dagger 17.94 mV zeta potential. A satisfactory prolidase encapsulation efficiency (43%) was obtained with a yield of the preparation process of similar to 55%. In vitro study of activity of prolidase, as free enzyme or released from chitosan nanoparticles, highlighted the ability of chitosan to stabilize the enzyme during all the steps of the preparation process and to modulate the enzyme activity up to 48 h.