Saints come into the fixture after an FA Cup victory over Championship side Coventry City. They needed extra time and a 112th-minute winner from former Spurs man Kyle Walker-Peters to secure a fifth-round spot.
Walker-Peters once more came up with the goods, netting an early goal for Hasenhüttl's men.
Spurs themselves enter the game in high spirits after a dominant 3-1 win over Brighton on Saturday evening.
The performance will have given Antonio Conte and his team confidence as they enter a busy period of fixtures.
There are no fresh injury concerns for the home side.
Eric Dier is rested once again as a precaution (thigh).
Conte told Spurs media:
"It’s not serious, but the problem is in the same place as last time, so, for this reason, we have to have a bit of patience and not take any risks.”
Southampton defender Lyanco will miss out on Wednesday evening's clash with a hamstring injury that will keep him out for up to 12 weeks. Young midfielder Nathan Tella will also be absent due to a recurring groin injury.
Saints will wait for a late assessment of on-loan Chelsea striker Armando Broja to assess his ability to feature.
Hasenhüttl said: "It’s possible that he is available for tomorrow. We don’t know yet."
Predicted Line Ups
Lloris; Royal, Sanchez, Romero, Davies, Reguilon; Bentancur, Hojbjerg; Lucas, Son, Kane.
Forster; Walker-Peters, Perraud, Bednarek, Salisu, Romeu, Ward-Prowse, S. Armstrong, Redmond, Adams, A. Armstrong.
Ones to watch
- Harry Kane
The England captain seemed back to his breath-taking best on Saturday evening.
Spurs fans will have been in dreamland watching him bend a trademark strike into the top corner.
His poachers finish not only capped off a strong Spurs display but also gave Kane his just rewards for his all-around link-up play and sharpness throughout.
The Saints defence will likely dread the thought of having to deal with Spurs' number ten.
The athletic defender dealt with almost everything that City could throw at him and he will by no means be ready to allow Kane and co an easy route to three points on Wednesday evening.
When Spurs visited the South-Coast on the 28th of December 2021, Saints managed to hold the Lilywhites to a 1-1 draw.
James Ward-Prowse's strike in the 25th minite was cancelled out by an emphatic Kane penalty as Southampton were reduced to 10-men.
A dubious offside VAR call denied Kane a winner and Conte's side were left frustrated.
Where to watch
The game will be televised on BT Sport 3 HD from 7PM (Kick-off 7.45 PM).