On a night when Barca paid tribute to the late Dutch great Johan Cruyff, the Catalan giants' 39-game unbeaten run came to a halt after goals from Karim Benzema and Cristiano Ronaldo erased Gerard Pique's opener.
“For me this game is history,” said Barca coach Luis Enrique, electing to turn the page rather than reflect on Saturday's loss.
“There is no pain. We came into the game on the back of many games without losing, but defeats can teach you things and the players know that.”
Barcelona have won the last six meetings with Atletico, but it was Los Rojiblancos who advanced when the two sides clashed at this stage of the 2013-14 competition - the only time Barca have failed to reach the semis in the past eight seasons.
Bayern Munich host Portuguese league leaders Benfica in the first leg with Pep Guardiola's side bidding to reach the last four for the fifth year running.
Bayern laboured to a 1-0 win at home to Eintracht Frankfurt as they remained five points clear of Borussia Dortmund in the Bundesliga, but skipper Philipp Lahm warned they would need to up their level against Benfica.
“After an international break it's often difficult. That's perfectly normal,” said Lahm.
“I think it's going to be a completely different match (on Tuesday). We'll be perfectly prepared. We need a better performance, but I'm not worrying about that.”
Bayern will be without Arjen Robben at the Allianz Arena, but the hosts should have France winger Kingsley Coman available after sitting out Saturday's victory with a leg injury.
Zlatan Ibrahimovic powered Paris Saint-Germain to a 4-1 win over Nice at the weekend and the French champions will again look to their Swedish star when they take on Manchester City at the Parc des Princes on Wednesday.
Paris reached the Champions League semi-finals in 1994-95, but City, despite their indifferent Premier League displays, are through to the last eight for the first time.
Manuel Pellegrini's side rediscovered some form at the weekend though with a 4-0 demolition of Bournemouth, and PSG midfielder Thiago Motta said it would take a complete team effort to overcome City.
“It's not Ibra against City. It's Paris Saint-Germain against City. In my head, I've never thought about taking to the pitch and winning a game all alone. It's impossible,” Motta told French daily L'Equipe.
“If we put pressure on him like that, and if he puts it on himself like that, we're not good. If we win, we'll win as a team.”
Marco Verratti is sidelined for PSG and Javier Pastore is doubtful, while captain Vincent Kompany is missing for City who are also sweating over the fitness of Joe Hart and Yaya Toure.
Six straight wins have reignited Real's season but the Champions League represents their only realistic chance of silverware this term.
Zinedine Zidane's men travel to Germany on Wednesday for a first-ever meeting with Wolfsburg and head into the tie as heavy favourites against a side embarking on their best run in Europe's premier competition.
However, Wolfsburg have won just three of their last 15 Bundesliga games and were soundly beaten 3-0 at Bayer Leverkusen on Friday.
“Things don't look good for us right now. You can't say we deserve to be higher in the standings than we are right now,” said Julian Draxler, with Wolfsburg languishing in eighth place in Germany.
The mood is far more upbeat in the Real camp having avenged November's 4-0 home drubbing at the hands of Barca, with Wales star Gareth Bale optimistic they can finish the season in style.
“We're feeling strong, confident now, and it's one final push until the end of the season,” said Bale.
“(Saturday's win) really does put us mentally in a strong position, we can beat anyone on our day and we showed that.
“Barcelona have been saying they're the best team and we beat them in their own backyard.” – AFP
Original source: Tricky Altetico test for Barca