Atletico Madrid striker Diego Costa, who was previously capped twice by his native Brazil, will make his first appearance for his adopted nation at the Vicente Calderon.
Having previously made his Brazil bow against the Azzurri last March, the Lagarto-born forward received Spanish nationality and made the controversial decision to switch international allegiances.
The Atletico Madrid striker, who boasts 21 La Liga goals this season, is now in line to represent la Roja for the first time, having been included in the defending world champions' squad to face Italy.
"He's an unusual case, because he had never been called up before and because he was born in Brazil," said Spain coach Vicente Del Bosque. "But he's a player who has been called up on the back of what he has achieved with his club and because we believe that he can help us. That's why we've called him up; for no other reason."
Italy can be expected to pose a stern test of Spain's FIFA World Cup credentials and the two sides are familiar with each other, having met four times inside the last three years.
With Del Bosque set to name his squad for the 2014 World Cup on May 25, the game against Italy could represent the last chance for Spain's fringe players to nail down a place.
Italy, meanwhile, has drawn both of its friendlies - against Germany and Nigeria - since qualifying for the finals and has fixtures with the Republic of Ireland and Luxembourg pencilled in before a World Cup opener against England in Manaus.
Roma midfielder Danielle De Rossi has been left out by Prandelli after throwing a punch at Mauro Icardi during Saturday's goalless draw with Inter.
Striker Mario Balotelli is also absent with a shoulder problem, but uncapped Torino striker Ciro Immobile could feature after scoring 13 Serie A goals this season.
Both sides were unbeaten in qualifying and topped their respective groups, although Spain's last two outings have been less than convincing.
La Roja narrowly beat Equitorial Guinea 2-1 in November before going down 1-0 to South Africa three days later.