This game was much more eventful than Saturday's 1-0 Premier League victory, with Graziano Pelle netting his second to make it 3-2 two minutes from time at the Britannia Stadium.
Saints looked in control at half-time at two up through Pelle and Shane Long's first for the club but Stoke levelled through Steven Nzonzi and Mame Biram Diouf.
However, moments after substitute Peter Crouch was sent off, Pelle grabbed his second to earn Southampton their ninth win in 10 games.
Both managers resisted the temptation to make wholesale changes, with 16 of the players on show having started at the weekend.
Mark Hughes gave Bojan Krkic a rare start, the former Barcelona forward appearing for the first time in nearly a month, while Saturday's goalscorer, Sadio Mane, missed out with a knock so Long came in for the visitors.
Despite the strong sides, there were plenty of empty seats at the Britannia Stadium, although more than 2,000 Southampton fans had made the trip.
And they were celebrating inside six minutes as Pelle notched his ninth of the season.
This was arguably his best, the Italian picking up the ball in the middle of the park and taking advantage of the space given to him by the Stoke defence to pick his spot just inside Asmir Begovic's post from 25 yards.
Stoke hit back with a spell of pressure but, like at St Mary's, were unable to create any real chances.
The closest they came was a 19th-minute cross from Jonathan Walters that Nathaniel Clyne got his head to just before Marko Arnautovic.
Saints certainly looked to have the greater class in attack and a flowing move ended with them doubling their lead on the half-hour mark.
Dusan Tadic played in Steven Davis on the right of the area and he pulled the ball back for Long to slot under Begovic.
Pelle should have made it three shortly afterwards but blazed over and salt was rubbed into Stoke's wounds when they thought they had scored only for Walters' shot to be ruled out for offside.
The striker was definitely standing beyond the last defender but the assistant referee waited until the ball was nestling in the net before raising his flag.
The Stoke players came out early for the second half and it took them only four minutes to halve the deficit.
Nzonzi picked the ball up in midfield from what looked a soft free-kick and drove towards the goal before unleashing a low shot into the corner behind the dive of Fraser Forster.
That certainly put an extra spring in Stoke's step and Bojan had the chance to really make his mark but he poked a tame shot wide after breaking into the area.
Southampton still looked very dangerous on the break and it would have been 3-1 in the 57th minute but for a fine tip-over from Begovic as Tadic found an unmarked Long.
The Stoke keeper then denied Victor Wanyama with his legs while Tadic also threatened after careless play from Arnautovic but saw his shot blocked.
Bojan was certainly heavily involved but his end product left a lot to be desired.
After blazing a shot well over the bar when others were better placed, the forward did well to rob Florin Gardos but could not pick out Diouf with a cross when an equaliser beckoned.
Failing to connect with Diouf's ball in front of goal proved Bojan's final contribution, with Crouch replacing him, while Charlie Adam came on for Geoff Cameron.
Adam's first act was to pick up a yellow card, Stoke's third of the night, for a foul on Morgan Schneiderlin, and the midfielder had to be dragged away by team-mates as he voiced his displeasure at the decision.
Adam made a better impression with a dangerous corner that Marc Wilson headed wide at the far post.
Five more corners followed in quick succession and, after near misses for Phil Bardsley and Crouch, Diouf scored.
Adam's cross was flicked on at the near post by Bardsley and Diouf made no mistake with a header for his first goal in more than a month.
It looked like extra-time beckoned but, having picked up a first yellow card moments earlier, Crouch was dismissed for fouling Davis and from the free-kick a mistake by Erik Pieters allowed Pelle to slam home the winner.
Source : PA
Source: PA