The League One side were found guilty by the Football Association (FA) of breaching FA Cup rules after naming defender Georg Margreitter in their starting line-up for the 1-0 win earlier this month.
According to the FA, Margreitter, on loan from Wolves, had not received the necessary written permission prior to the fixture on December 6.
Chesterfield were charged following the original victory, but remained in the third-round draw while an investigation took place and Paul Cook's side will now have to play the second-round fixture again.
"Following an FA Cup sub-committee hearing at Wembley Stadium on Monday, Chesterfield have been ordered to replay their FA Cup second round proper fixture against Milton Keynes Dons," an FA statement read.
"Chesterfield had admitted a breach of FA Cup Rule 15(j), after fielding an ineligible player, namely Georg Margreitter, in their 1-0 win at MK Dons on Saturday 6 December 2014.
"After considering submissions made by Chesterfield in relation to the charge, the committee has ordered the tie to be replayed at MK Dons on the earliest available date as decided by The FA."
The winner of the replay will travel to either Scunthorpe United or Worcester City in the third round.
