Mauricio 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.