Luke McGee was handed his first appearance for more than nine months in the Group D loss at the Keepmoat Stadium.
He was the sole senior player to feature, though, with academy players making up the rest of Mark Kelly’s side.
Joe Wright scored the only goal of the game for Rovers in the 18th minute.
Kelly heaped praise on McGee professionalism and the experience he added to a youthful team.
And the academy chief took plenty of positives from Pompey’s performance.
‘It was a really good display from the kids against a strong, powerful Doncaster side,’ Kelly told the club’s website.
‘We got opened up in the early stages, although then settled into the game and we were pleased with the boys, who were giving a lot away in terms of age.
‘They had quite a few boys with first-team experience, but after getting to grips with our shape, we played some excellent football at times.
‘We were brave with what we were trying to and it was just that cutting edge in the final third that was missing.
‘There were a few good opportunities. Josh Flint hit their lad on the line, Bradley Lethbridge sent one over the bar and there were other bits and pieces.
'Compared to what we were up against, the boys handled themselves really well and showed a high standard of football.
‘Luke McGee was the skipper for the night and did a very professional job. He was great with the back four and the rest of the team.’
Doncaster started the better team, with Alex Kiwomya and Elliot Walker going close in the early stages.
Rovers made their pressure count and took the lead on 18 minutes when Wright headed home a free-kick.
Pompey made a good response after falling behind, though.
Wright was forced into defensive duties a minute later when he cleared Josh Flint’s effort off the line before Gerard Storey sent a free-kick over the bar.
Despite playing his first match since December, McGee – who was keen to depart Fratton Park during the summer – showed few signs of rustiness.
In the 23rd minute, the keeper tipped a Donny triallist’s effort over and then made a similar save from Danny Amos seven minutes later.
The hosts fashioned a couple of chances early in the second half, while Bradley Lethbridge fired off target from the edge of the box for the Blues on 61 minutes.
McGee made his third fine stop of the evening with 11 minutes remaining, thwarting Amos’ low drive before Will Longbottom headed the rebound wide.
Pompey are also joined by Aston Villa under-23s and Exeter in Group D.
The reserves are in Central League Cup action against AFC Bournemouth on Tuesday, October 1 at Baffins Milton Rovers’ PMC Stadium (7pm).