Cheltenham will monitor Jeff Goulding ahead of the visit of the Dons.
Goulding has a cut ankle which has seen him miss some training but the striker is expected to train on Friday.
Midfielder Sam Deering and forwards Chris Zebroski and Shaun Harrad, who was on the scoresheet along with Goulding in a 2-1 friendly win at Solihull Moors on Tuesday night, are pushing for their competitive debuts.
Manager Mark Yates is mulling over a possible deal for midfielder Jordan Hibbert and goalkeeper Connor Roberts as he looks to add a number two as cover for Scott Brown but it remains to be seen whether anything will be finalised ahead of the first-round tie.
Cheltenham were beaten 4-1 at Whaddon Road by the Dons at this stage of the competition last season after going in at half-time on level terms.
"I expect MK Dons to have more quality than they did last season," Yates told the club's official website. "We improved dramatically after playing them last season and the game will be a good guide for us. I suppose there is a little less pressure with it not being a league game but we want to beat them. Ultimately we will use it for our fitness levels ahead of the new league campaign."
MK Dons will wait to see whether a quartet of players are fit enough for the trip.
Defenders Mathias Kouo-Doumbe and Jon Otsemobor, midfielder Daniel Powell and striker Jabo Ibehre were absent as Karl Robinson's side lost 1-0 to a late Fulham goal in a pre-season friendly at stadium.mk on Tuesday night.
Doumbe and Ibehre were nursing injuries carried over from a previous warm-up game at Kettering while Powell was ill and summer signing Otsemobor was also rested as a precaution.
Robinson is hopeful they will all be fit for the first-round tie on this weekend.
Alan Smith and Ryan Lowe, the other two new arrivals at the Milton Keynes club over the close season, will be pushing for starting places.