The Spireites lost for the first time at home under James Rowe as Sutton United took a 1-0 result away from the Technique Stadium.
Four changes were made from the weekend as Fraser Kerr, Joel Taylor, Curtis Weston and Jack Clarke came in for Will Evans, Alex Whittle, Martin Smith and Liam Mandeville.
The visitors looked to get off to the perfect start through Donovan Wilson. The forward made his way through on goal and his shot was straight at Grant Smith.
Jak McCourt almost opened the scoring at the other end when his shot from outside the box deflected off Ben Goodliffe, but Dean Bouzanis collected the ball before being wrong-footed.
Chesterfield continued to pressure the Sutton defence; Gavin Gunning rose highest to meet McCourt’s corner and his bullet header flew just wide of the post.
Rowe was forced to make a substitution in the 22nd minute. Kerr, who went down with an knock in the opening ten minutes, hit the deck once more and was replaced by Whittle.
The deadlock was broken with just over ten minutes of the first half remaining. Wilson once again found a way through on goal and rolled his shot past Smith and into the bottom left corner.
Omar Buigel almost doubled the lead soon after; he made his way into the penalty area via the left byline before his low shot crashed off the post and the ball was cleared.
During the half-time break, Nathan Tyson warmed up and was sent on in place of Tom Whelan.
The first chance of the second half fell to Weston who controlled the ball on the edge of the area and saw his half-volley deflected inches wide of the post.
Sutton continued to look for the goal to give them breathing space. Harry Beautyman was allowed to shoot from just inside the penalty are, but Smith was equal to the effort.
With just over 20 minutes to go, Gavin Gunning was forced off with a hamstring injury and he was replaced by Haydn Hollis.
The Spireites kept going to find an equaliser and a way back into the game. Weston hammered a shot towards goal, which Bouzanis somehow got his fingertips to and tipped over the bar.
Unfortunately, Chesterfield couldn’t find a precious goal to get back on level terms.
Chesterfield: G Smith, Carline, Kerr (Whittle 22), Gunning (Hollis 68), Maguire, Taylor, McCourt, Weston, Whelan (Tyson 46), Clarke, Asante.
Unused Substitutes: Mandeville & Dinanga.