Rovers' Premier League Youth Cup campaign got off to a disappointing start following an opening defeat against Sheffield United on Friday.
Damien Johnson included first team duo Lewis Travis and Paul Downing in the side against the Blades for the tie played at Stocksbridge Park Steels.
Goals from Jordan Hallam, Oliver Greaves and Simeon Oure put the hosts in control, before Rovers pulled one back through Stefan Mols.
The early stages produced little of note, but it was the Blades who broke the deadlock when Greaves and Hallam combined and the latter produced a cool finish by lifting the ball over Charlie Albinson after being played through.
Soon after and the lead was doubled; this time it was Greaves, who finished off a nicely worked corner routine by smashing a strike from the edge of the box and into the corner via a deflection.
It was a bitter blow for Rovers, who were struggling to create too many clear cut chances in the first half and went in at the break with a mountain to climb.
Into the second half, it was the Blades who added to the tally as Oure sneaked away from his man and slotted calmly into the bottom corner of the net to ensure there was further daylight between United and the visitors.
Rovers did pull one back late on through substitute Mols, who hammered home from inside the box when the ball fell his way.
Rovers are next in action on Friday 19th October when they head south to take on Brighton & Hove Albion in Premier League 2, kick-off 7pm in Sussex.
Sheffield United U23s: Amissah, Gordon, Bryan (Mallon 38), Broadbent, Cantrill, York, Bennett, Greaves (Weaver 90), Oure (Parkhouse 83), Hallam, Boyes.
Unused substitutes: S. Kelly, Neal.
Rovers: Albinson, Platt, Evans, Travis, Downing, Grayson (c), Carter (Vale, 66), Lyons, Simmonds (Candlin, 81), Butterworth, Buckley (Mols, 55).
Unused substitutes: Winterbottom, Saadi.