Luis Enrique decided to leave the Chelsea defender out of his 23-man selection after he played 87 minutes of the defeat to Switzerland last week, which knocked La Roja off top spot of Group A2.
Real Madrid right-back Dani Carvajal is set to take Azpilicueta’s place while Barcelona’s Eric Garcia has also been left out.
The 33-year-old has been a regular under Luis Enrique and a run of 14 consecutive Spain appearances ended in June, his longest run in the national team.
Chelsea boss Graham Potter will no doubt be happy to see Azpilicueta rested however, having started him in defence for his debut in the dugout against RB Salzburg earlier this month.
The Blues travel to Crystal Palace on Saturday before games against AC Milan and Wolves. Both Christian Pulisic and Edouard Mendy are set to be in action for their countries this week.
Portugal have left Wolves winger Pedro Neto and Benfica’s Goncalo Ramos out of their matchday squad as Joao Cancelo and Joao Felix return from suspension and injury, respectively.
You can follow Portugal vs Spain LIVE on Standard Sport tonight…