Fernando Santos's side are also preparing for fixtures against France and Sweden, and the Portugal manager has several selection dilemmas when it comes to Wednesday's exhibition match with their neighbouring nation.
Portugal and Spain have not locked horns since the 2018 World Cup, where the spoils were shared in a six-goal thriller in the group stage, and Portugal ran out 4-0 winners when the two heavyweights last met in a friendly 10 years ago.
Here, Sports Mole looks at how Portugal could line up for their midweek showdown with Spain.
With two important Nations League clashes to come for the reigning champions, Santos is expected to give a number of international first-timers the chance to impress in Wednesday's friendly.
One man who is set to make his debut for the national team is Braga left-back Nuno Sequeira - the 30-year-old has been called up to the squad for the first time following the news of Napoli's coronavirus cluster - which forced Mario Rui to withdraw.
On the opposite side of defence, new Wolverhampton Wanderers recruit Nelson Semedo is vying for a start over Joao Cancelo, and Semedo is joined in the squad by a number of his Wolves teammates in Rui Patricio, Ruben Neves, Joao Moutinho and Daniel Podence.
The latter has suffered a minor injury setback but nevertheless turned out for the West Midlands outfit against Fulham at the weekend, and Podence is in contention to earn his first cap for Portugal on the right flank.
Cristiano Ronaldo may be given the night off as Santos looks to avoid any injury concerns before the Nations League fixtures, which could see Liverpool's Diogo Jota handed a start on the left-hand side of a front three, with Joao Felix battling Andre Silva to spearhead the Portugal attack.
In the middle of the park, William Carvalho has earned a recall to the national setup and could feature in the holding role, with Premier League duo Moutinho and Bruno Fernandes ahead of him, although Danilo Pereira, who joined Paris Saint-Germain on loan from Porto on deadline day, is another option.