Albion were knocked out of the Carabao Cup by Southampton after Charlie Austin headed home a late winner at the Amex.
Austin nodded home the only goal two minutes from time when he met fellow sub Nathan Redmond's left-wing cross.
Brighton were unable to find an equaliser to take the game to penalties and Saints progressed into the third round.
Albion made ten changes from the 1-0 Premier League defeat at Liverpool on Saturday, with only midfielder Yves Bissouma keeping his place in the starting 11.
Goalkeeper David Button and youngsters Will Collar, Ben Barclay and Viktor Gyokeres all made their Seagulls debuts, while club record signing Alireza Jahanbakhsh started for the first time.
Captain Bruno, Florin Andone, Jose Izquierdo and Lewis Dunk were all still ruled out.
Southampton also made ten changes from their late 2-1 defeat at home to Leicester on Saturday. Winger Mohamed Elyounoussi was the only player to keep his place in the starting line-up.
The visitors went close twice in quick succession after eight minutes. Elyounoussi's point-blank range header from James Ward-Prowse's corner was superbly kept out by Button and Maya Yoshida's follow-up was blocked by Barclay.
Albion then had a couple of openings at the other end but Bissouma and Gyokeres both fired over from the edge of the penalty area after driving runs forward.
Jahanbakhsh then shot just over from 20 yards on 19 minutes, before Saints midfielder Stuart Armstrong curled an effort wide two minutes later.
Button pushed away a sharp low shot from Gabbiadini on 36 minutes and Armstrong sent an effort just over on the stroke of half-time.
Elyounoussi drilled a shot straight at Button in the first real action of the second half on 52 minutes as clear openings remained few and far between.
Button was called into action again to keep out Gabbiadini's low strike on 57 minutes, before Albion went close at the other end a minute later. The impressive Bissouma delivered a cross which was met by Gyokeres but Angus Gunn dived to his left to push away the young winger's header.
Albion keeper Button denied Gabbiadini again on 64 minutes as the Southampton striker's 20-yard free kick was pushed to safety.
At the other end, Beram Kayal had a close-range shot blocked on 72 minutes before Gyokeres's follow-up was saved by Gunn at the near post.
Saints substitute Sam Gallagher turned the ball wide from Steven Davis's cross three minutes later and then headed at Button shortly afterwards.
Gallagher had another opening on 78 minutes but Button kept out his low strike after neat work down the right.
The visitors continued to press and had a great chance to go ahead on 83 minutes as Button miskicked and Gallagher squared the ball to Charlie Austin, whose low shot was cleared off the line by Bernardo.
Southampton went ahead with just two minutes to go when Austin met fellow sub Redmond's cross and headed home from eight yards to send Saints through to the third round.
Brighton: Button; Collar, Barclay, Bernardo, Suttner; Jahanbakhsh, Kayal (Connolly 90), Gross (Propper 62), Bissouma, Gyokeres; Locadia. Subs: Steele, Balogun, Montoya, Stephens, Knockaert.
Southampton: Gunn, Stephens, Yoshida, Bednarek, Targett, Romeu (Gallagher 70), Ward-Prowse, Elyounoussi, Armstrong (Redmond 80), Davis, Gabbiadini (Austin 80). Subs: Lewis, Vestergaard, Hoedt, McQueen.