The youth team went down to a 4-3 defeat at Salford City on Tuesday afternoon after taking a 2-0 lead inside 20 minutes.
The turning point came when the Spireites lost keeper Kurtis England, who was sent off in the first half.
Chesterfield played some good football early on and goals from Javanne Smith and Josh Turner put them in control of the game.
The hosts pulled a goal back following a corner and then came the incident which saw the Spireites reduced to ten men. England raced out of his area, committed a foul and was shown a red card.
From the resulting free-kick, Salford equalised to make it 2-2 going into the break.
Salford scored twice more to go 4-2 up before Smith reduced the arrears late on.
Reflecting on the game, academy manager Matt Whitehouse said: "It was a really positive opening 20/25 minutes when we played some really good football and created a number of good chances.
"The sending off really affected our confidence and we never recovered from that.
"Javanne Smith worked hard throughout the game and posed a threat."
The young Spireites are next in action on Thursday night when they face their counterparts from York City in a National League Cup tie.
Spireites: England, Garwood (Brookes), Netherwood, Geeves, Barlow, Wild, Fuertado, Clogg, Smith, Turner (Gibson), Hargreaves (Wharton)