Holmes was taken off after 70 minutes of the weekend game and left the ground on crutches after falling awkwardly on his left knee.
The player has been assessed by club physio Anders Braastad and will have the injury scanned on Tuesday.
“It’s not great news and it’s a blow because he’s been outstanding for us,” said manager Chris Wilder.
“It was an innocuous situation, he just planted his foot.
“There was a lot of swelling on his knee after the game and he will definitely be out for Tuesday.”
Ivan Toney will be available for the game against Carlisle despite suffering a heavy knock early in his substitute appearance on Saturday.
The blow limited Toney’s ability to run, but he is not expected to miss any training in the build-up to the visit of the Cumbrians to Sixfields.
Joel Byrom returns from a two-game suspension, but Kaid Mohamed (foot), Ian Morris (knee), Ben Tozer (knee) and Marc Richards (Achilles) are all out.