Cody McDonald's goal early in the second half saw the Spireites beaten 1-0 by table-topping Gillingham on Saturday.
With Sam Hird out through suspension, Liam Cooper captained the side while Jack Lester and Danny Whitaker both dropped down to the bench. Richard Brindley came into the side to make his full league debut at right-back with Craig Clay and Conor Townsend also handed places in the starting line-up.
Gillingham's Charlie Lee threatened inside the opened minute of the game before his scuffed shot went wide.
Tommy Lee then pulled off a great save, down low to his right, to thwart Chris Whelpdale.
Chesterfield's first effort of the match came from Jay O'Shea whose angled shot went wide.
McDonald received a good ball from Steven Gregory and raced into the area, forcing Neal Trotman to make a last-ditch tackle to clear the danger. Leon Legge then headed over as the visitors continued to press.
Marc Richards put a first-time effort over from the edge of the area after O'Shea's cross was headed down by Drew Talbot.
Trotman tried his luck from distance but the ball went well wide and Gillingham's Joe Martin shot wide at the end of the half.
McDonald scored five minutes after the re-start, meeting Myles Weston's corner with a close-range header.
After Townsend fired just over, Gills substitute Deon Burton had a free-kick deflected wide and Lee saved comfortably from his namesake.
A double substitution saw Alex Henshall, signed on loan from Manchester City yesterday, and Jack Lester join the action in the 53rd minute. They replaced O'Shea and Clay.
Henshall was soon involved, finding Lester whose effort was blocked.
Cooper did well to cut out Whelpdale's cross as the visitors looked to catch Chesterfield on the break.
Legge, who posed an aerial threat all afternoon, had a header cleared off the line and Burton's shot was deflected wide.
Lester, off-balance, had a shot saved as the Spireites threw men forward in search of an equaliser.
The 81st minute introduction of another new signing, Jordan Clark, in place of Trotman saw the formation switched to 3-5-2.
Chesterfield had plenty of possession towards the end without seriously testing Stuart Nelson in the Gillingham goal.
Chesterfield: Lee, Talbot, Trotman (Clark 81), Cooper, Smith, Brindley, Togwell, Clay (Lester 53), Townsend, O'Shea (Henshall 53), Richards Unused subs: Barrington, Randall, Whitaker, Hazel.
Gillingham: Nelson, Fish (Burton 28), Martin, Legge, Barrett, Whelpdale, Gregory, Robinson, Lee, McDonald (Davies 59), Weston. Unused subs: Forecast, Richardson, Dack, Williams, Birchall.
Referee: Robert Madley