Atletico holds a narrow advantage after scoring an away goal in the 1-1 first leg result at the Camp Nou, and can expect raucous support from a fan base that has tasted much success in the Europa League of late, but has not reached the final four among Europe's elite since 1974.
Atletico is clinging to the hope that striker Diego Costa will overcome a hamstring injury picked up in the first leg, while playmaker Arda Turan is also on the mend.
Barcelona is vying to reach the semifinals of the Europe's top-tier competition for the seventh straight season.
Here are five things to know about Wednesday's matchup between the Spanish league rivals:
COME ON COSTA
Atletico Madrid striker Diego Costa has been quite possibly the key to Atletico's improbable season, which sees it sitting top of the Spanish league - one point ahead of Barcelona - with just six games to play. But the Spain striker's availability could prove crucial to advancing deeper into the competition for the first time since 1974. Midfielder Arda Turan could return after also missing Saturday's 1-0 win over Villarreal.
Atletico coach Diego Simeone said Tuesday that Costa would only play if 100 percent, even suggesting little-used Adrian Lopez could start in attack despite the return of Raul Garcia from a ban.
''Costa is to us what Cristiano (Ronaldo) is to Madrid or (Lionel) Messi to Barcelona. But I don't know what percent he's at for a return,'' Simeone said before the final training which could determine Costa's participation. ''A player is only useful if he's a minimum 90 percent (fit). A player who is 50 percent is of no use to us.''
Barcelona midfielder Xavi Hernandez wants his team to score quickly and place the pressure on Atletico.
''Then we can see how they react,'' Xavi said.
Atletico, alongside Real Madrid, is the only unbeaten team left in the competition.
Lionel Messi, who has scored 20 career goals against Atletico, may be the best option but has yet to break through and beat Thibaut Courtois in four meetings this season as Atletico's league-best defense has been the most effective team in a long period at shutting down the Argentina forward. Neymar has scored both of Barcelona's goals against Atletico this season as the teams have drawn in all four meetings.
Andres Iniesta will make his 500th appearance for Barcelona against Atletico, nearly 12 years after making his first-team debut. Iniesta's first appearance also came in the Champions League - against FC Brugges - and he has helped Barcelona win 21 trophies besides scoring the winning goal in Spain's World Cup triumph against the Netherlands in 2010. The Spain international has been especially important down the stretch for the Spanish champion with defender Gerard Pique (hip) and goalkeeper Victor Valdes (knee) out.
''Our season is on the line,'' Iniesta, 29, said.
PRESSURE, WHAT PRESSURE
Barcelona may have more experience when it comes to reaching this stage of Europe's top tournament, but Atletico captain Gabi Fernandez said Tuesday there was no pressure on his team. Atletico has been battling Barcelona and Madrid in the race for the Spanish league title, including the season-long doubts and questions over whether it could maintain its unexpected title charge to the close.
''We are in the peak moment of our lives. No one is tired,'' Gabi said. ''We've earned the right to be here.''
Home field advantage always carries sway in a two-legged tie, an aspect Atletico is counting on utilizing at the Vicente Calderon. Atletico's boisterous fans certainly gave the team a lift during a 4-1 victory over AC Milan in the return leg of its last 16 victory, and the team expects no less against Barcelona. ''The atmosphere has rumbled and that empowers you, gives you energy, gives you strength to compete even harder,'' Simeone said. ''When you ask us why we are Atletico, that's why. We're passionate, hard workers, and humble, and if we need to suffer (to win), we suffer.''