Oxford will be without midfield duo Jake Forster-Caskey and Lee Cox as they look to cause an upset against the Blades after the pair returned to their parent clubs.
Manager Chris Wilder, a boyhood Blades fan and former player, will have to fill the holes left in his squad with Forster-Caskey having gone back to Brighton and Lee Cox's return to Swindon despite Cox and United being keen to arrange a longer stay.
Daniel Boateng is also returning to his parent club Arsenal having made just five appearances for the U's.
Michael Dubbery was one of Oxford's best performers in the win over Cheltenham having replaced the ill Michael Raynes - leaving Wilder with a selection dilemma at the heart of his defence.
Meanwhile striker James Constable serves the first match of his three-game ban for his second red card of the season, picked up in the win against AFC Wimbledon.
Sheffield United manager Danny Wilson is expected to include new signing Jamie Murphy in his squad.
Attacking player Murphy, 23, completed his permanent move from Motherwell for an undisclosed fee on Thursday and could make his debut for the club.
Defender Harry Maguire is also available again after sitting out the New Year's Day draw at Doncaster through suspension.
Wilson, who has no new injury or suspension worries, is likely to retain recent signing Danny Higginbotham in his back four following Neill Collins' cheekbone fracture, while teenagers Terry Kennedy and Elliott Whitehouse, who both made their debuts on the New Year's Day, and another 19-year-old, Joe Ironside, are hoping to keep their places in the side.
Goalkeeper Mark Howard (thigh) returned to training this week, but is hoping to feature in a couple of development games before being considered for his long-awaited first-team return.
The Blades beat Oxford 3-0 at Bramall Lane in the first round of last year's competition.