La Roja's preparations for this summer's competition have been difficult, with Busquets and then Diego Llorente testing positive for coronavirus, but the latter has now recorded two negative tests.
There are a number of unknowns when it comes to team selection, with relatively few of the current squad actually guaranteed positions in the first XI.
Aymeric Laporte and Pau Torres could line up as the two centre-backs, while Marcos Llorente and Jordi Alba should make up the back four despite pressure from Cesar Azpilicueta and Jose Gaya.
In midfield, Rodri could start due to Busquets's absence, while Thiago Alcantara and Koke are likely to be handed positions in the XI for Monday's Group E contest.
Gerard Moreno enjoyed an excellent 2020-21 campaign for Villarreal, meanwhile, and he is expected to feature in a wide area, with Ferran Torres also being tipped to make the starting side.
Alvaro Morata is the leading goalscorer in the squad with 19 - 11 more than anyone else - and the 28-year-old should be given the nod at centre-forward, with the likes of Adama Traore, Mikel Oyarzabal, Pablo Sarabia and Dani Olmo set to start on the bench.
Spain possible starting lineup: Simon; M Llorente, Laporte, P Torres, Alba; Thiago, Rodri, Koke; F Torres, Morata, G Moreno