Having lost their Checkatrade opener to high-flying Peterborough United in August, Southampton's youngsters will look to get their Trophy campaign back on track against Cambridge United on Tuesday evening.
One of 16 clubs invited to field an under-21 side in the competition, Saints progressed to the knockout stages last year, but will have their work cut out against League Two's 12th-placed side tonight.
The U's enter the tie in mixed form, but are unbeaten at home since the beginning of August, and drew one apiece with the group's other competitors, Northampton Town, in the first round of fixtures.
Previewing the tie, coach Radhi Jaïdi said: "We've spoken to the players about games coming thick and fast, and they are important games.
"We're playing a senior team at their stadium, so it'll be challenging for our boys. They need to be ready to bounce back and react following Friday's game (a 2-1 defeat to Watford for Saints' Under-23s side).
"It is a great opportunity; how are the younger boys going to deal with the challenge? The ones who step up will send a good message.
"It's both encouraging and intimidating to play in a stadium, you have to deal with the crowds as well as the opposition, and the psychological side of the game.
"I'm expecting it to be physical and direct, the ball will be in the air, and obviously if you give them time and space they will play."