Tendayi Darikwa gave the home side an early lead, but headers from Marc Laird and Anthony Straker put Southend ahead before former Chesterfield midfielder Kevan Hurst sealed victory late on.
Chesterfield were in front in the fourth minute when Gary Roberts crossed low to the far post where Darikwa fired past Southend keeper Dan Bentley, but Southend equalised eight minutes later when Laird headed in Ben Coker's corner.
The home side lost Roberts through injury in the 16th minute and were behind 60 seconds later when Hurst crossed from the right for Straker to plant a back post header past Chesterfield keeper Tommy Lee.
Ollie Banks had a shot deflected over Bentley's goal in the 43rd minute and Drew Talbot headed against the top of the Southend bar in the 52nd minute, but Southend had a great chance to settle the tie in the 79th minute.
Sam Hird barged over Barry Corr to concede a penalty, but the striker's spot-kick was superbly saved low to his left by Lee, before Hurst finished it in the 85th minute from 12 yards after great work by Straker.