The Spireites bowed out of the Vertu Trophy at the Round of 32 stage after losing 5-1 to Doncaster Rovers at the Eco-Power Stadium.
Nine changes were made to the side from the weekend, with Chey Dunkley and Liam Mandeville staying in the starting XI.
Luke Butterfield made his first start for the club after returning from a broken foot; Gunner Elliott also made his first senior start for the club.
Vontae Daley-Campbell had to make a last-stitch challenge in the first minute to deny Glenn Middleton.
James Berry and Elliott had a couple of early chances. The former’s curling effort was saved by Thimothée Lo-Tutala, and the latter’s header drifted just wide of the mark.
However, in the 15th minute, Doncaster took the lead through Billy Sharp’s long-ranger effort, which flew beyond Ryan Boot.
Damola Ajayi added a second for the home side five minutes later. His shot from the edge of the area wrong-footed Boot and found the back of the net.
Just after the half-hour mark, Jack Senior increased the host’s lead. He latched onto Ajayi’s through ball before slotting the ball into the goal.
Tom Naylor and Dylan Duffy were brough on after the break, replacing Butterfield and Elliott.
The final third saw very little action for the first two-thirds of the second period as Chesterfield looked to find a way back into the game.
In the 75th minute, Daley-Campbell found substitute Lee Bonis with a wicked cross, which the striker directed into the back of the net, via the post.
Sharp added his second of the night, two minutes later. He touched down Tom Nixon’s cross before turning and firing beyond Boot.
The forward claimed his hat-trick a minute into added time as he saw his deflected effort looped into the goal.
Chesterfield: Boot, Daley-Campbell, Grimes, Dunkley, Lewis, Butterfield (Naylor 46), Mandeville (Gordon 76), Darcy (Cook 86), Dickson, Berry (Bonis 73), Elliott (Duffy 46).
Subs not used: Tanton, Whitney.
