Hodgson told a news conference he was keen to experiment against both the European champions and France in another friendly on Tuesday, when Rooney would be playing, but refused to reveal his starting lineup for the Spain game.
With Rooney and Vardy missing, Hodgson is likely to deploy Harry Kane and Ross Barkley up front for the clash in Alicante, the first meeting between the pair since England beat La Roja 1-0 in a friendly in London in late 2011.
"Jeremy Vardy is struggling with a knock he picked up on Saturday," Hodgson said.
"We're monitoring it, we're still working on it but it looks unlikely at this moment in time that he'll be able to play tomorrow.
"Wayne's with me today (at the news conference) because he's the captain of our team and is the most important figure as a result but he actually won't start the game tomorrow.
"That's for the very simple reason ... that we're looking at various combinations amongst that group of four players and Wayne is in actual fact pencilled in to play the game against France on Tuesday."
Rooney, who has netted a record 50 goals in his 107 appearances for England, said he understood the need for Hodgson to look at other players before the Euro 2016 finals in France starting in June.
"It's important especially in these games that the manager has a chance to see as many players as he can," Rooney said.
"Obviously as a player you want to play but you have to understand that there's bigger things to consider in terms of tournament football.
"Hopefully the lads who come in can do well and give the manager something to think about."
(Writing by Iain Rogers, editing by Pritha Sarkar)
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