Pochettino made an unsurprising nine changes to the side that managed a 1-1 draw with Sunderland on Saturday, leaving out his big-name buys Dani Osvaldo, Victor Wanyama and Dejan Lovren.
Instead, there were chances for fringe players such as Lloyd Isgrove and Emmanuel Mayuka, and they made the most of their opportunities in a comfortable victory over the Sky Bet Championship's bottom club.
Midfielder Isgrove, making his first start and only his second appearance for Southampton - with his debut in this competition last October - set up the opener in the 26th minute for Steven Davis.
Four minutes after the break Jay Rodriguez, who had a two-month loan spell with the Tykes in early 2010, doubled the visitors' advantage with a stunning 18-yard curled effort beyond debutant Mike Pollitt.
Veteran Pollitt was only recruited hours ahead of the game, with boss David Flitcroft signing the 41-year-old on a month's loan from Wigan.
For Pollitt, it was his first match since January when he played the first half of Wigan's 1-0 FA Cup third-round replay victory at Bournemouth, and only his third outing in two and a half years.
Barnsley at least gave themselves a lifeline in the 54th minute via skipper Stephen Dawson's thumping 22-yard effort for his first goal since December.
But all hope of a comeback was ended in the 66th minute when Zambia international Mayuka netted his first for the Saints following his move from Swiss club Young Boys last summer.
Mayuka was then fouled in the dying stages by Jimmy McNulty for Davis to add a fourth from the spot, before Gaston Ramirez slotted home the fifth from close range in injury time.
Pollitt, associated with 15 clubs during a career stretching back to 1990 when he started out as a trainee with Manchester United, was thrown in at the deep end, replacing Luke Steele.
Steele had shipped 12 goals in his opening four league games this season, including a handful on Saturday in a 5-2 thrashing at Blackburn.
Pollitt, hardly at fault for any of the goals, could easily have been left picking the ball out of his net after just 54 seconds as Barnsley's obviously shaky defence this season was cruelly exposed.
There must have been a sigh of relief in seeing a 15-yard left-foot shot from Ramirez flash inches past his left-hand post after the Uruguayan midfielder had been played in behind the back four.
Four minutes later and Ramirez, last summer's £12million club record buy before the £15million capture of Osvaldo just over a week ago, had the home rearguard all at sea again with a whipped free-kick from wide on the left that Rodriguez flicked wide.
With the visitors comfortably in control, Pollitt was called on for the first time in the 13th minute, pushing away a long-range effort from Daniel Fox that Rodriguez failed to hook home on the rebound.
Barnsley, far from overawed but undoubtedly lacking the quality of their opponents, at least registered one decent first-half chance.
From a long clearance by Pollitt, Chris O'Grady flicked the ball on into the path of Marcus Pedersen, the Norwegian striker signed on loan from Vitesse Arnhem.
Pedersen showed a turn of pace to elude his markers, but from just inside the area flashed a 15-yard effort across Kelvin Davis and narrowly beyond his right-hand post.
It was a moment of light relief in the Saints half for the home side as the south-coast club made their breakthrough in the 26th minute.
Isgrove was the instigator with a ball into Davis, who proceeded to thump it home from 15 yards into the top right-hand corner.
Pollitt's body later managed to block a swerving 25-yard drive from Rodriguez before Jacob Mellis almost levelled late on with a rasping effort that fizzed past the right-hand post.
Just four minutes after the interval, however, the match appeared to be put beyond Barnsley's reach when Rodriguez swept an 18-yard curler beyond Pollitt into the bottom left-hand corner.
Three minutes later and the hosts should have been dead and buried, only for Rodriguez to smack an edge-of-the-area drive against the base of the right-hand post.
Instead, Barnsley hauled themselves into the game courtesy of Dawson, only to fall apart soon after as initially Mayuka flashed a cracking drive past Pollitt.
Davis' penalty and Ramirez's effort put the gloss on the win, and the scoreline was unfair on Barnsley, but the Saints go marching on.