La Roja plays friendlies against Albania and Iceland during the upcoming international break, and Luis Enrique has replaced De Gea with another goalkeeper from the Premier League, Brentford's David Raya.
From the previous squad for the final FIFA World Cup qualifiers in November, in defence, Inigo Martinez and Jose Luis Gaya are replaced by Diego Llorente and Marcos Alonso, while Marcos Llorente and Pedri come in as midfield options in place of Busquets and Mikel Merino.
Raul de Tomas and Ferran Torres are selected among the forwards, with no place for Rodrigo, Pablo Fornals or Brahim Diaz.
Speaking after the squad announcement, Luis Enrique said that Busquets's omission was merely to keep him fresh. The 33-year-old has played 39 games in all competitions for Barca this season.
"In the case of Busquets, it is a personal decision," he confirmed. "He is the player who accumulates the most minutes and I want to have the best version of the captain for the games in June, and I have decided to rest him.
"He is having a spectacular season."
The Spain head coach also said the two upcoming fixtures should not be treated like friendlies, and challenged his squad to show its "ambition" and "hunger" as the FIFA World Cup at the end of the year draws closer.
He added: "Since November, the wait has been long [to play again]. They are not friendlies for the players nor for us because the World Cup is close. It will help me to see the level of the players, their ambition, their hunger."
Unai Simon, Robert Sanchez, David Raya; Marcos Alonso, Jordi Alba, Aymeric Laporte, Pau Torres, Eric Garcia, Diego Llorente, Cesar Azpilicueta, Dani Carvajal; Rodri, Koke, Pedri, Marcos Llorente, Gavi, Carlos Soler; Dani Olmo, Pablo Sarabia, Alvaro Morata, Raul de Tomas, Ferran Torres, Yeremy Pino.