A Paul McGinn own goal and a double from Ivan Toney gave the home side a 3-0 lead before Tom Anderson pulled a goal back in stoppage time.
The result means the Spireites will be playing League Two football next season.
The Spireites made two changes from the loss to Southend last week as Rai Simons and David Faupala came in for Dan Jones and the suspended Dion Donohue. Gary Caldwell reverted to a 4-3-3 formation as he looked to get his side back on track.
Joe Rowley was fast becoming a vital cog in the Spireites side and the 17-year-old flicked the ball around his marker on the halfway line before charging forward as he looked to spark his team’s first attack; Rowley then curled the ball forward for Dennis, but the striker was beaten to it by Conor Townsend.
Scunthorpe had the first real opportunity of the match, Scott Wiseman dragged the ball across the six-yard box and Rowley cleared, but it only went as far as Jamie Ness on the edge of the area who powered it wide.
Neal Bishop wasted a similar chance moments later, as Scunthorpe began to take a foothold on the game, Thorsten Stuckmann grasped the midfielder’s strike which had taken a deflection on its way through the area.
The Spireites stopper was then forced into another save by Bishop when the midfielder leapt highest at a corner and directed a header into the left corner but Stuckmann pushed wide.
Scunthorpe took the lead in the 21st minute from another corner, the ball was launched into the box and McGinn couldn’t react fast enough to prevent the ball from bouncing off him and into his own net.
In search of an instant equaliser Dan Gardner charged down the left-wing and drove a low ball across the box that Scunthorpe cleared for a corner.
Caldwell was forced into an early change as Reece Mitchell replaced the injured Simons and the winger almost made an instant impact, thumping a half volley at Joe Anyon who parried well.
The hosts attempted to clear their lines but Connor Dimaio latched onto the rebound and curled a 20-yard effort just past the right post.
Scunthorpe doubled their advantage on the counter, Bishop crossed the ball for Toney (37) who rose above Stuckmann and headed over the stopper and into the empty net.
Mitchell battled with Bishop on the attack and attempted to lay the ball for Rowley, the young mid-fielder snatched it from the pair and thundered an effort at goal, but it was blocked and hit clear.
Scunthorpe returned from the break in an equally attacking fashion and Paddy Madden thought he had notched a third early on. The striker had been set one-on-one with Stuckmann by Toney, but the stopper dove to his left to palm wide.
Madden thought he had gotten the better of the German ‘keeper minutes later from a corner, but his effort crashed against the crossbar and bounced off the line.
Dawson received the ball in the area, cut onto his right and fired a shot into the top right corner, but once again Stuckmann was there to acrobatically tip it wide.
Mitchell found space on the left and curled the ball into Dennis, the forward leapt above his marker and nodded at Anyon but instead it hit the side netting.
Stuckmann had been keeping the Spireites in the game, but was finally beaten during a collision with Anderson and Toney (73), the latter poked the ball beneath the two and it dribbled into the net.
Gardner cut inside on the edge of the area and played the ball to Dennis, the striker drilled a low ef-fort at Anyon but the stopper held comfortably.
Chesterfield earned themselves a consolation in the dying embers of the game, Anderson (90+) took advantage of a lapse in concentration from the hosts and nodded past Anyon.
Scunthorpe United: Anyon, Wiseman, Wallace, Mirfin, Dawson (C), Madden (van Veen), Morris, Bishop, Townsend, Toney (Davies), Ness (Mantom).
Subs not used: Watson (gk), Toffolo, Holmes, Sutton.
Chesterfield: Stuckmann, McGinn, Hird (C), Gardner, Faupala (Jones), Dimaio, Dennis, Simons (Mitchell), Anderson, Maguire, Rowley (Wakefield).
Subs not used: Fulton (gk), Angel, Ebanks-Blakel, Grimshaw.