Goals from Conor Chaplin and Carlton Morris were enough for the Tykes to seal an impressive win in London as they put an end to the Bees' 21 game unbeaten run.
Their hit and miss form this campaign has meant they have slipped down the table and are now seven points off the play-off places.
The Tykes will be without Aapo Halme, Ben Williams and Liam Kitching as they all remain sidelined with injuries.
Top scorer Cauley Woodrow is likely to replace Morris in the lone striker position despite impressing with a goal against Brentford.
Orlando City loanee, Daryl Dike, looks set for his second start after a very positive league debut.
Tony Mowbray's side has been riddled with injuries this season, as Bradley Johnson, Daniel Ayala, Scott Wharton, Lewis Holtby and Joe Rankin-Costello are all set to miss the tie.
Amari'i Bell and Barry Douglas will battle it out for a starting place at left-back as they have been rotated in and out of the side all season.
Walton; Andersen, Helik, Sollbauer; Brittain, Palmer, Mowatt, Styles; Chaplin, Woodrow, Dike.
The hosts tend to operate in the 3-4-3 formation with the wide midfielders dropping in, turning it into a back five when they are defending.
Kaminski; Bell, Brantwaite, Lenihan, Nyambe; Davenport, Travis; Elliott, Dack, Rothwell; Armstrong.
Mowbray has stuck with his 4-2-3-1 formation for the majority of the season. He utilises a flat back four, two holding midfielders, two wingers, a number ten and a lone striker.
Ones to watch
Woodrow is Barnsley's top scorer this season with nine goals. He also has four assists to his name and has averaged a goal every 225 minutes so far this season.
His eye for goal is only one part of his game but he offers a lot more for the Yorkshire side. He has a wonderful ability to hold the ball up and bring others into play around him. His first touch and eye for a pass in the final third is a real danger.
As many lone strikers do, Woodrow has the legs to put meaningful pressure on opposition defenders with his energy and work rate always being second to none.
The 26-year-old can strike to ball with both feet and has possession of a fierce drive both from distance and in the penalty area.
He has created 29 chances so far this season as well as making 25 key passes. He could be the catalyst to Barnsley's success if he performs well.
Adam Armstrong has been Blackburn's go-to man this season having scored 18 goals and notching three assists.
Armstrong is the second top scorer in the SkyBet Championship and has averaged a goal every 129 minutes.
His clinical finishing has been important to the Rovers as he has single-handedly won them games and gained them points this campaign.
The 24-year-old possesses a venomous eye for goal and is the definition of a fox in the box with all 18 goals having come within the penalty area.
He too can strike off both feet having scored seven with his left foot and nine with his right.
The Englishman has lightning pace and is at his best when he is played in behind defences. He is certainly one that Barnsley need to keep quiet if they are to get anything from this game.
Out of the last nine Championship meetings between these two sides, Barnsley have won three, Blackburn have won five and there has been one draw.
When they met earlier on this season, Blackburn were victorious after goals from Armstrong and Sam Gallagher sealed a 2-1 win for Mowbray's men.
The last time Blackburn did the double over Barnsley in the Championship was right back in 2012.
Last time out:
Where to watch
The game kicks off at 19:45 BST on Wednesday 17 February 2021.
For both sets of fans, the game can be watched on the Sky Sports Football channel by pressing the red button and choosing this game.