The reserves went down to a 3-1 defeat at Bradford City on Monday afternoon.
The Spireites trailed by a goal to nil at the break with Jacob Hazel, playing against his home-town team, having a first-half effort saved.
Striker Kristian Ramsey-Dickson, who is training with Chesterfield, came on as a half-time replacement for Mark Fereday.
On-loan winger Alex Henshall equalised around the hour-mark with a left-foot volley from around 20 yards after a corner had been headed out.
Bradford regained the lead after some poor marking at a corner, allowing the home player to tap in.
A re-shuffle saw Jordan Slack slotting in at right-back after coming on in place of Hazel with Jonathan Wafula pushed further up.
The hosts added a third goal when a left-wing cross was dropped by Spireites keeper Josh Barrington at the feet of a Bradford player who turned the ball home.
Ramsey-Dickson fired in a good effort which was turned over the bar and Mark Randall also forced the home keeper to make a good save.
Tim Whittaker, Jordan Clark and Danny Whitaker also had chances to score in the second period.
Coach John Dungworth said: "It was disappointing to lose like that after conceding some sloppy goals. We played some good football without being clinical in front of goal."
Chesterfield: Barrington, Wafula, McNicholas, Randall, Broadhead, Whittaker, Fereday (Ramsey-Dickson), Clark, Hazel (Slack), Whitaker, Henshall. Unused subs: Mason, Scully, Hewitt.
The reserves are at home to Rotherham on Wednesday afternoon (kick-off 2pm).